Tumor-initiating stem cells fine-tune the plasticity of neutrophils to sculpt a protective niche

Abstract

The heterogeneous nature of tumor-associated neutrophils (TANs) has been recognized, but how different cell states of TANs emerge, evolve, distribute, and impact cancer immunotherapy efficacy remain elusive. Using single-cell RNA sequencing, spatial transcriptomics, and genetic manipulations, we show that anti-PDL1 + CD40 agonist immunotherapy can induce interferon responses in TANs, allowing them to regain anti-tumor activities in squamous cell carcinomas (SCCs). In contrast, TANs residing at the tumor-stroma interface can preserve their immune-suppressive state. Importantly, we identify a group of SOX2^High tumor-initiating stem cells (tSCs) at the tumor-stroma interface that upregulate fatty acid desaturase 1 (Fads1) to produce arachidonic acid (AA). This tSC-specific pathway enhances the prostaglandin E₂ (PGE₂) signaling in TANs, which can disrupt the interferon response and prevent the interferon-induced anti-tumor functions in TANs. By fine-tuning the plasticity of neutrophils, tSCs shape neutrophil heterogeneity and sculpt a protective micro-niche to survive from immunotherapy and drive cancer relapse.

Keywords: Fads1; Sox2; arachidonic acid; cancer relapse; immunotherapy; interferon response; prostaglandin E2; squamous cell carcinoma; tumor-associated neutrophils; tumor-initiaiting stem cells.

Figure 1.. Immunotherapy induces distinct responses in different neutrophil subpopulations.

A. Schematic of experimental procedures and growth curve of spontaneous skin SCCs after anti-PDL1 + CD40 agonist immunotherapy treatment. n = 5 in each group. B. UMAP showing the immune cell types identified by scRNA-seq in skin SCCs. C. UMAP showing the TAN subpopulation clustering in skin SCCs. D. UMAP showing the signature and immune suppressive genes expressed in various TAN clusters. E. UMAP and stacked bar chart showing the changes in the composition of TAN subpopulations induced by the anti-PDL1 + CD40 agonist treatment. F. Chord diagram showing the upregulated pathways induced in each cluster of TANs after the anti-PDL1 + CD40 agonist treatment. G. Bubble heatmap showing transcripts of various immune stimulatory genes induced in cluster 1 – 3 (T1 to T3), and immune suppressive genes induced in cluster 4 (T4) after anti-PDL1 + CD40 agonist treatment. Two-way ANOVA and Sidak’s multiple comparisons tests were used in (A), and results in (A) are presented as mean ± SEM. **p < 0.01. See also Figure S1 and Table S1

Figure 2.. The interferon responses triggered by immunotherapy can restore the anti-tumor functions in TANs

A. Experimental scheme and flow cytometry quantification of T cell responses when TANs were depleted before immunotherapy treatment. n = 5 for Cre⁻ and n = 6 for Cre⁺ group. B. Experimental scheme and flow cytometry quantification of T cell responses when TANs were depleted during the anti-PDL1 + CD40 agonist treatment. n = 15 for Cre⁻ and n = 12 for Cre⁺ group C. Growth curve of SCC tumors with or without TAN depletion during immunotherapy treatment. n = 18 for Cre⁺ group and n = 10 for Cre⁻ group. D. Representative flow cytometry histogram showing the upregulation of markers associated with anti-tumor activity (e.g. LY6E or MHCII), but downregulation of immune-suppressive markers (e.g. Siglec-F) in TANs after anti-PDL1 + CD40 agonist treatment. Note that these changes cannot be detected in neutrophils in other tissues (bone marrow or spleen) from the same tumor-bearing mice. E. Experimental scheme, flow cytometry quantification of LY6E and MHCII expression (left) and qPCR (right) quantification of interferon stimulated gene (Gbp2 and Ifit3) expression in lineage traced pre-existing TANs (Tomato⁺) before or after the immunotherapy treatment. Gbp2 and Ifit3 expression levels were first normalized to 18S rRNA and then further normalized to the average value of the naive group. n = 4 for naive and n = 6 for treated group in flow cytometry, n = 3 for each group in qPCR. F to H. Flow cytometry quantification of (F) MHCII expression in neutrophils or (G) IFNγ production in CD4⁺ T cells, and (H) the growth of SCC tumors before and after immunotherapy treatment when both IFNα and IFNγ receptors are deleted specifically in neutrophils (Ifnar1/Ifngr1 dKO). n = 20 control group, n = 10 for the dKO group. Graphs from A to H show representative results from one of the three repeats for each experiment and was presented as mean ± SEM. Student’s t tests (A, B and E), Mann-Whitney U Tests (D), and two-way ANOVA and Sidak’s multiple comparisons tests (C, F, G and H) were used for statistical comparison. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, non-significant. See also Figures S2 and S3

Figure 3.. Spatial distribution of distinct neutrophil responses during immunotherapy treatment

A. Experimental scheme and representative IF images of skin SCCs collected for selecting different ROIs either within the tumor (Tu) or in the stroma (St) for spatial transcriptomic analysis. B. Heatmap showing scaled expression of anti-tumor immunity-related genes in neutrophils located at the tumor-stroma interface or in stroma before and after anti-PDL1 + CD40 agonist treatment. C. Chord diagram showing the down-regulated pathways in neutrophils located at the tumor-stroma interface compared to the neutrophils in stroma during immunotherapy treatment. D and E. Representative IF images and quantifications of (D) Gbp2⁺ or (E) MHCII⁺ neutrophils among all the MPO⁺ cells located at the tumor-stroma interface (arrow) or in stroma (arrowhead) after immunotherapy treatment. The tumor-stroma interface was labelled with white dotted line. n = 30 in D, n = 36 in E. Scale bars: 50 μm. Mann-Whitney U Tests were used for statistical comparison in D and E, and data are presented as mean ± SEM. *p < 0.05, **p < 0.01. See also Figure S4

Figure 4.. Sox2 amplification allows SCC cells to dampen interferon responses in TANs.

A. MA plot showing the up-regulated TFs in sorted ITGA6⁺ CD80⁺ tSCs from skin SCCs compared to differentiated cancer cells. Genes with significant differential expression (adjusted p-value < 0.05) are shown as pink dots. B. Experimental scheme and representative flow cytometry plots quantifying upregulation of CD14 and LY6E in TANs isolated from tumors formed by SCC cells individually amplifying various tSC-specific TFs. C. Flow cytometry quantification of the MHCII expression in TANs from SCC tumor cells with or without amplifying Sox2, before and after anti-PDL1 + CD40 agonist treatment. n = 8 in SOX2^Low group and n = 7 in SOX2^High group. D. Heatmap showing the differentially expressed genes in neutrophils isolated from SCC tumors with (SOX2^High) or without (SOX2^Low) amplifying Sox2, before (Naive) and after (Treated) anti-PDL1 + CD40 agonist treatment. E. Leading edge plots showing the downregulated pathways in neutrophils isolated from SOX2^High SCC tumors compared to the neutrophils isolated from the SOX2^Low tumors after the anti-PDL1 + CD40 agonist treatment. F. Quantitative PCR measuring the expression of interferon stimulated genes (Gbp2 and Ifit3) in neutrophils isolated from naive SOX2^Low or SOX2^High tumors following in vitro IFNα + IFNγ treatment for 4 hr. Expression levels of Gbp2 and Ifit3 were first normalized to 18S rRNA and then further normalized to the average value of the SOX2^High group. n = 8 in each group. G and H. Experimental scheme, representative IF images and quantification of (G) the percentage of MHCII⁺ TANs among all the MPO⁺ cells or (H) the number of CD4⁺ T cell-neutrophil interactions per imaged area in SOX2^High (arrow) or SOX2^Low regions (arrowhead) when SOX2^Low and SOX2^High cells were mixed at 7:3 ratio for grafting. Scale bars: 50 μm. n = 40 in G, n = 30 in H. I. Experimental scheme and flow cytometry quantification of IL2 production in OTII CD4⁺ T cells after co-culture with OVA-loaded neutrophils isolated from SCC tumors formed by SOX2^Low or SOX2^High cells. n = 6 in each group. J and K. Representative IF images and quantification of (J) the HLA-DR⁺ TANs among all the MPO⁺ cells or (K) the number of CD4⁺ T cells per imaged area in SOX2^High (arrow) or SOX2^Low regions (arrowhead) in HNSCCs samples collected from immunotherapy-treated patients. The tumor-stroma interface was labelled with white dotted line. Scale bars: 100 μm. n = 30 in J, n = 50 in K. Graphs in C, F, G, H, I, J and K show representative results from one of the three repeats for each experiment and results are presented as mean ± SEM. Two-way ANOVA and Sidak’s multiple comparisons tests (C), Student’s t tests (F, G, H, I, J and K) were used for statistical comparison. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, non-significant. See also Figures S4-S6 and Tables S2-S4.

Figure 5.. SOX2 is critical for tSCs to shape neutrophil cell states, block neutrophil-T cell interactions, exclude T cells and drive cancer relapse.

A. Model schematics and growth curve of spontaneous skin SCCs with or without ablating Sox2 in basal epithelium before the anti-PDL1 + CD40 agonist treatment. n = 12 in control, n = 14 in Sox2 cKO. B. UMAP and stacked bar chart showing the changes in the composition of neutrophil subpopulations induced by the anti-PDL1 + CD40 agonist treatment when Sox2 is ablated in tSCs. C. UMAP showing expression level of various immune stimulatory genes expressed in different TAN clusters in WT or Sox2 cKO SCCs with or without anti-PDL1 + CD40 agonist treatment. D to F. Representative IF images and quantification of (D) the Gbp2⁺ or (E) the MHCII⁺ neutrophils among all the MPO⁺ cells, and (F) the number of interactions between neutrophils and CD4⁺ T cells in the imaged area in WT (arrow) or Sox2 cKO (arrowhead) SCC tumors after anti-PDL1 + CD40 agonist treatment. The tumor boarder was labelled with white dotted line. Scale bars: 50 μm. n = 60 in D, n = 100 in E, n = 90 in F. G and H. Growth of SCC tumors formed by (G) SOX2^High or SOX2^Low SCC cells with or without anti-PDL1 + CD40 agonist treatment, or by (H) SOX2^High cells with or without neutrophil depletion after anti-PDL1 + CD40 agonist treatment. n = 8 in each group in G and n = 10 in each group in H. I and J. Representative IF images and quantification of (I) the number of CD4⁺ T cells in the imaged tSC-enriched regions or (J) the percentage of dead (active Caspase3⁺) SOX2⁺ tSCs in spontaneous SCCs with (arrow) or without (arrowhead) neutrophil depletion after anti-PDL1 + CD40 agonist treatment. The tumor boarder was labelled with white dotted line. Scale bars: 100 μm. n = 60 in I, n = 100 in J. Representative results in A, D to F, I and J are from one of the three repeats, and presented as mean ± SEM. Mann-Whitney U Tests (D to F, I and J) and Two-way ANOVA and Sidak’s multiple comparisons tests (A, G and H) were used for statistical comparison. *p < 0.05; **p < 0.01; ****p < 0.0001; ns, non-significant. See also Figure S7.

Figure 6.. SOX2 activates FADS1 to block the interferon-induced anti-tumor functions in TANs.

A. Heatmap showing the differentially expressed genes in Sox2 OE SCC cells compared to Sox2 KO cells. B. IGV image showing the SOX2 or control antibody CUT & RUN. C to E. Flow cytometry quantification of the (C) LY6E, (D) MHCII and (E) Siglec-F expression in TANs in immunotherapy-treated SCC tumors formed by SOX2^High cells with or without silencing Fads1. n = 5 in each group. F. Quantitative PCR measuring Ifit3 expression in TANs isolated from naive SCC tumors formed by SOX2^High cells with or without silencing Fads1 following treatment with IFNα + IFNγ in vitro for 4 hr. Expression levels of Ifit3 were first normalized to 18S rRNA and then further normalized to the average value of the group without Fads1 silencing. n = 9 in each group G to I. Flow cytometry quantification of the (G) LY6E, (H) Siglec-F, and (I) MHCII expression in TANs in immunotherapy-treated SCC tumors formed by SCC cells with or without amplifying Fads1. n = 6 in each group. J. Quantitative PCR measuring Gbp2 expression in TANs isolated from naive SCC tumors with or without amplifying Fads1 following treatment with IFNα + IFNγ in vitro for 4 hr. Expression levels of Gbp2 were first normalized to 18S rRNA and then further normalized to the average value of the vector control group. n = 12 in each group. K to N. Flow cytometry quantification of CD4⁺ T cell infiltration (K) and IFNγ production (L), as well as CD8⁺ T cell infiltration (M) and IFNγ production (N) in immunotherapy-treated SCC tumor cells formed by SCC cells with or without amplifying Fads1. n = 6 in each group. Bar graphs from C to N show representative results from one of the three repeats for each experiment and are presented as mean ± SEM. Student’s t tests (C to J) and one-way ANOVA followed by Tukey’s multiple-comparison tests (K to N) were used for statistical comparison. *p < 0.05; **p < 0.01.

Figure 7.. AA induces PGE₂ signaling to disrupt the interferon response in TANs.

A. Quantitative mass spectrometry measurement of AA in TIF extracted from naive SCC tumors formed by SOX2^Low or SOX2^High tumors. Relative AA levels were normalized to the average amount in SOX2^Low tumors. n = 6 in each group. B. Experimental scheme and quantitative PCR measuring the Gbp2 and Ifit3 expression in TANs isolated from SCC tumors with or without intratumoral injection of AA for 5 days. Isolated neutrophils were treated with IFNα + IFNγ in vitro for 4 hrs to measure their interferon responsive capacity. Expression levels of Gbp2 and Ifit3 were first normalized to 18S rRNA and then further normalized to the average value of vehicle control. n = 5 in each group. C and D. Quantitative PCR measuring the (C) Gbp2 and (D) Ifit3 expression in cultured primary neutrophils treated with vehicle or different AA metabolites for 48 hrs followed by IFNα + IFNγ treatment for 4 hr to measure their interferon responsive capacity. Expression levels of Gbp2 and Ifit3 were first normalized to 18S rRNA and then normalized to the average value of the vehicle control group. n = 4 in each group. E. Quantitative PCR measuring the Ptgs2 expression in TANs isolated from naive SCC tumors formed by SOX2^Low or SOX2^High SCC cells. Expression levels of Ptgs2 were first normalized to 18S rRNA and then normalized to the average value of the SOX2^Low group. n = 8 in each group. F. Western blots probing the phosphorylation of JAK1, JAK2 and STAT1 in cultured primary neutrophils after treatment with vehicle or different AA metabolites for 48 hrs followed by IFNα + IFNγ in vitro treatment for 30 mins to quantify the impacts of PGE₂ on the interferon signaling transduction in neutrophils. Actin was used as a loading control. G to I. Flow cytometry quantification of the (G) MHCII and (H) LY6E on LY6G⁺ TANs or (I) the LY6G- myeloid cells in SCC tumors formed by SOX2^High SCC cells on control or the Ptger2/Ptger4 myeloid cell-specific dKO mice. n = 8 in control and n = 6 in dKO group. J. Growth of SOX2^High or SOX2^Low SCC tumors following combinational treatment of Cox2 inhibitors (Celecoxib or Aspirin) and immunotherapy. n = 10 in each group. Graphs show pooled results (A) or representative results (B to J) from one of the three repeats for each experiment and are presented as mean ± SEM. Student’s t tests (A, B, E, G, H, I), one-way ANOVA followed by Tukey’s multiple-comparison tests (D and E), and Two-way ANOVA and Sidak’s multiple comparisons tests (J) were used for statistical comparison. *p < 0.05; **p < 0.01; ***p < 0.001.