Spatially distinct tumor immune microenvironments stratify triple-negative breast cancers

Abstract

Understanding the tumor immune microenvironment (TIME) promises to be key for optimal cancer therapy, especially in triple-negative breast cancer (TNBC). Integrating spatial resolution of immune cells with laser capture microdissection gene expression profiles, we defined distinct TIME stratification in TNBC, with implications for current therapies including immune checkpoint blockade. TNBCs with an immunoreactive microenvironment exhibited tumoral infiltration of granzyme B+CD8+ T cells (GzmB+CD8+ T cells), a type 1 IFN signature, and elevated expression of multiple immune inhibitory molecules including indoleamine 2,3-dioxygenase (IDO) and programmed cell death ligand 1 (PD-L1), and resulted in good outcomes. An "immune-cold" microenvironment with an absence of tumoral CD8+ T cells was defined by elevated expression of the immunosuppressive marker B7-H4, signatures of fibrotic stroma, and poor outcomes. A distinct poor-outcome immunomodulatory microenvironment, hitherto poorly characterized, exhibited stromal restriction of CD8+ T cells, stromal expression of PD-L1, and enrichment for signatures of cholesterol biosynthesis. Metasignatures defining these TIME subtypes allowed us to stratify TNBCs, predict outcomes, and identify potential therapeutic targets for TNBC.

Keywords: Breast cancer; Expression profiling; Oncology.

Figure 1. Therapy-naive TNBC tumors are classified into subtypes on the basis of distinct spatial localization of CD8⁺ T cells.

(A) Representative images of CD8⁺ T cell staining at the tumor margins (top panels, dotted lines) and in the tumor core (bottom panels) (n = 38). Scale bars: 100 μm. (B) Quantification of CD8⁺ T cell densities at the tumor margins (marCD8) and in the tumor core (corCD8) (n = 38). (C) Comparison of strCD8 with sTILs and epiCD8 with iTILs (n = 38). Data were analyzed using Spearman’s correlation. (D) Working model of TNBC grouping based on CD8⁺ T cell localization. Black, green, blue, and red represent ID, MR, SR, and FI tumors, respectively. marCD8, corCD8, strCD8, and epiCD8 are the CD8⁺ T cell densities in the tumor margin, core, and stromal and epithelial compartments, respectively. Data represent the mean ± SEM.

Figure 2. TIME subtypes compared with PAM50 and Lehmann breast cancer subtype stratifications.

(A) Comparison of CD8⁺ T cell grouping (TIME subtypes) with PAM50 molecular subtyping of our TNBC cohort (n = 37). (B–D) Comparison of CD8⁺ T cell grouping (TIME subtypes) with Lehmann molecular subtyping of TNBC (n = 37), showing enrichment of the mesenchymal and immunomodulatory subtypes in corCD8^lo (ID + MR) and corCD8^hi (SR + FI), respectively. Data were analyzed by Fisher’s exact t test.

Figure 3. CD8⁺ T cell localization–derived metasignature methodology.

(A) Analysis workflow for metasignatures and associated biological processes discovery in our data set (n = 37) and validation of the external data set (n = 578). (B) Heatmap on the left shows clustering of the pathway scores, determined according to a positive or inverse correlation with CD8⁺ T cell density in the tumor core (corCD8), and identifies 4 corCD8 metasignatures (corCD8 MSig) in whole-tumor gene expression for all TNBC tumors (n = 37). Heatmap on the right shows clustering of the pathway scores, determined according to a positive or inverse correlation with CD8⁺ T cell density in the tumor epithelium (epiCD8), and identifies 6 epiCD8 metasignatures (epiCD8 MSig). epiCD8 MSigs were generated from whole-tumor gene expression in tumors showing CD8⁺ T cell infiltration into the tumor core (corCD8^hi, n = 22, SR and FI tumors).

Figure 4. CD8⁺ T cell localization–derived metasignatures identify distinct biological processes.

(A and B) Cellular pathways positively or inversely correlated with (A) CD8⁺ T cell density in the core (corCD8) of all TNBC tumors (n = 37) and (B) with CD8⁺ T cell density in the epithelium (epiCD8) of TNBC corCD8^hi (SR and FI) tumors (n = 22). Data were analyzed using Spearman’s correlation. Cellular pathway score FDR values are represented in whole tumor (black), tumor stroma (gray), and tumor epithelium (light gray). The top 5 significant pathways per metasignature are represented (except for corCD8 Msig1, for which the top 10 pathways are represented to reflect the dominance of the MSig). MODY, maturity-onset diabetes of the young; CTL, cytotoxic T lymphocyte; ESC, embryonic stem cell; GGPP, geranylgeranyl pyrophosphate; GVHD, graft versus host disease. Pathway names have been abbreviated; full names of the pathways can be found in Supplemental Tables 2 and 3.

Figure 5. Tumors with poor infiltration of CD8⁺ T cells are enriched for fibrotic foci.

(A) Representative image of fibrotic focus in H&E-stained images. Original image size: 7 × 10 mm; enlarged inset size: 0.5 × 0.5 mm. Dotted line identifies the fibrotic focus area. (B) Fibrotic focus presence represented as a 2D plot of marCD8 over corCD8 densities (n = 38). (C) Fibrotic focus presence was enriched in corCD8^lo compared with corCD8^hi tumors (n = 38). Data were analyzed using the Fisher’s exact test. (D) corCD8 was higher in tumors that did not contain a fibrotic focus (n = 38). Data were analyzed using the Mann-Whitney U test. Data represent the mean ± SEM.

Figure 6. Fully inflamed TNBC are associated with a proinflammatory TIME.

(A and B) Heatmap depicting the expression of genes associated with a type 1 IFN response and cytotoxic activity in the tumor stroma (A) and epithelium (B) (n = 22). (C) Representative images showing a higher number of GzmB⁺ CD8⁺ T cells in FI tumor epithelium compared with numbers in SR tumor. Pan-cytokeratin (Pan-CK) staining (pink) identifies tumor cells, and DAPI (blue) identifies nuclei. White squares outline the position of the zoomed area in the stromal region for SR tumor and the epithelial region for FI tumor. Scale bars: 50 μm (merge, CD8, and GzmB) and 5 μm (enlarged insets showing CD8 and GzmB colocalization). n = 22. (D) Quantification of GzmB⁺CD8⁺ T cells in the tumor core, tumor stroma, and tumor epithelium (n = 32). Data were analyzed using the Kruskal-Wallis test. Data represent the mean ± SEM. (E) GzmB⁺CD8⁺ T cell density in tumor epithelium was positively correlated with epiCD8 (n = 20). Data were analyzed using Spearman’s correlation. Green, blue, and red dots represent MR, SR, and FI tumors, respectively.

Figure 7. FI TNBC tumors are infiltrated with proinflammatory macrophages.

(A) Representative images of CD68⁺CD206^– and CD68⁺CD206⁺ macrophages show proinflammatory CD68⁺CD206^– accumulation in FI tumors (n = 15). White squares indicate the position of the zoomed-in stromal regions for MR and SR tumors and epithelial region for the FI tumor. Pan-CK (pink) identifies tumor cells and DAPI (blue) identifies cell nuclei. Scale bars: 50 μm (merge, CD68, and CD206) and 5 μm (enlarged insets showing CD68 and CD206 colocalization). (B and C) Quantification of CD68⁺CD206^– (B) and CD68⁺CD206⁺ (C) macrophages in each tissue compartment across groups (n = 15). Green, blue, and red dots represent MR, SR, and FI tumors, respectively. Data were analyzed using the Kruskal-Wallis test and represent the mean ± SEM.

Figure 8. SR TNBC tumors are defined by a cholesterol gene expression signature and a distinct TIME.

(A) Heatmap depicting the expression of genes of the SPP of cholesterol biosynthesis in bulk tumor from SR and FI tumors (n = 22). (B) Signature score of genes depicted in (A). Data were analyzed with Spearman’s correlation. (C) Signature score of ISGs repressed by SREBP2 showing decreased expression in SR versus FI tumors (n = 22). Data were analyzed with Spearman’s correlation. (D) Representative IHF images showing the presence of IL-17–producing cells in the tumor stroma of SR and FI tumors (n = 22). Blue, DAPI; pink, pan-CK; yellow, IL-17F. White squares represent the zoomed position in the images. Scale bars: 50 μm (merge) and 20 μm (enlarged insets). (E and F) Density of IL-17–producing cells (E) and neutrophils (F) across SR and FI tumors (n = 10; 5 patients with the lowest and 5 patients with the highest epiCD8, respectively, for SR and FI tumors). Data were analyzed by Mann-Whitney U test and represent the mean ± SEM.

Figure 9. TIME metasignatures show prognostic value in the external TNBC cohort.

(A) Analysis pipeline showing (a) Discovery of metasignatures in a 2-step process (corCD8 and then epiCD8 stratification); (b) prediction in our training set; and (c) validation of the external set. (B) Cohen’s κ statistics measuring the prediction accuracy of each metasignature and combinations. (C) Recurrence-free survival curves using the identified combinations of corCD8 MSig (up; n = 337) and epiCD8 MSig (down; n = 196). For the corCD8 MSig immune^hi fibrosis^lo versus immune^lo fibrosis^hi, the HR is 0.63 (0.456, 0.887), P = 0.006. For the epiCD8 MSig IFN^hi cholesterol^lo versus IFN^lo cholesterol^hi, the HR is 0.52 (0.330, 0.843), P = 0.01. The log-rank P value is shown on the plots. (D) Working model of TNBC stratification into immune subgroups on the basis of the metasignatures identified.

Figure 10. TIME TNBC subtypes express distinct markers of immune suppression.

(A) Heatmap depicting the expression of classical immunosuppressive genes in tumor stroma and epithelium for each patient (n = 38). (B) Signature scores for the immunosuppressive gene list from A (n = 38). Data were analyzed using the Kruskal-Wallis test and represent the mean ± SEM. (C) VTCN1 (B7-H4) and CD274 (PD-L1) gene expression was inversely correlated (n = 37). Data were analyzed using Spearman’s correlation.

Figure 11. TIME TNBC subtypes display mutual exclusion and distinct localization of PD-L1 and B7-H4.

(A) Representative images of PD-L1 (green), B7-H4 (pink), and DAPI (blue) IHF-stained sections. Scale bars: 10 μm and 20 μm (enlarged insets). (B) Quantification of staining in the tumor epithelial compartment for B7-H4 (pink) and PD-L1 (green) for each patient (n = 35). (C) B7-H4 and PD-L1 quantification after IHF show an inverse correlation in the tumor epithelial compartment. This excludes tumors with less than 1% staining in the epithelial compartment for both markers. Data were analyzed using Spearman’s correlation.

Figure 12. Schematic of TIME TNBC subtype stratification.

Poorly infiltrated tumors (ID and MR) are characterized by signatures of fibrosis, enrichment of fibrotic foci, and expression of the immune checkpoint B7-H4. Tumors that display significant infiltration of CD8+ T cells into the tumor core but that showaccumulation specifically in the tumor stroma, display signatures of cholesterol and infiltration of IL-17–producing cells and neutrophils. These tumors also display stromal expression of the immune checkpoint PD-L1. Tumors of the FI TIME subtype are consistently positive for MHC-I, whereas MHC-1 loss is observed in tumors from other TIME subtypes. Tumors with significant CD8+ T cell infiltration in the tumor epithelium are characterized by type 1 IFN signatures as well as activated CD8⁺ T cell (GzmB⁺) and PD-L1 expression in the tumor epithelial compartment. Scale bar: 100 μm.