Senescent CAFs Mediate Immunosuppression and Drive Breast Cancer Progression

Abstract

The tumor microenvironment (TME) profoundly influences tumorigenesis, with gene expression in the breast TME capable of predicting clinical outcomes. The TME is complex and includes distinct cancer-associated fibroblast (CAF) subtypes whose contribution to tumorigenesis remains unclear. Here, we identify a subset of myofibroblast CAFs (myCAF) that are senescent (senCAF) in mouse and human breast tumors. Utilizing the MMTV-PyMT;INK-ATTAC (INK) mouse model, we found that senCAF-secreted extracellular matrix specifically limits natural killer (NK) cell cytotoxicity to promote tumor growth. Genetic or pharmacologic senCAF elimination unleashes NK cell killing, restricting tumor growth. Finally, we show that senCAFs are present in HER2+, ER+, and triple-negative breast cancer and in ductal carcinoma in situ (DCIS) where they predict tumor recurrence. Together, these findings demonstrate that senCAFs are potently tumor promoting and raise the possibility that targeting them by senolytic therapy could restrain breast cancer development. Significance: senCAFs limit NK cell-mediated killing, thereby contributing to breast cancer progression. Thus, targeting senCAFs could be a clinically viable approach to limit tumor progression. See related article by Belle et al., p. 1324.

Figure 1.. Senescent CAFs are present in human breast tumors.

(A) UMAP of stromal cells from 26 human breast cancer samples of ER+ (n=11), HER2+ (n=5), and TNBC (n=10) cases. (B) Violin plots of CAF signature markers: vCAF—MCAM+, ACTA2+, DES+, NOTCH3+; myCAF—PDGFRA+, ACTA2+, THBS2+, POSTN+; iCAF—PDGFRA+, CXCLl12+, COL14A1+, ALDH1A1+. (C) Feature plot and violin plot for the expression of CDKN2A (p16) in selected stromal cell types. (D) Feature plot and violin plot for the expression of CDKN2B (p15) in selected stromal cell types. (E) Dot plot displaying gene expression parameters including percentage of cells (size of black dots) and expression levels (colored scale bar) of a designated gene within a specific population (y-axis). Features include senescence markers (CDKN2A (p16) and CDKN2B (p15) and SASP factors (GLB1, COL1A1, COL12A1, MMP10, MMP13, CXCL9, TNFSF11, CD276, and TGFB1). (F) Gene set enrichment analyses of the myCAF population compared to all other CAF populations show enrichment in pathways including extracellular matrix organization, collagen assembly, and Fridman senescence. Normalized Enrichment Score (NES), p-value, and False Discovery Rate (FDR) are shown in each plot.

Figure 2.. p16 expression is present in myCAFs and the presence of p16+ myCAFs correlates with DCIS recurrence.

(A) mIHC were performed on 51 human breast cancer tumor tissue samples using p16 (red), pan-cytokeratin (PanCK; cyan), PDGFRa (magenta), alpha-smooth muscle actin (aSMA; green), and COL14A1 (yellow) specific antibodies and nuclei were counterstained with hematoxylin and shown as dark blue. Individual stains were pseudo colored as indicated and then merged (first panel). myCAFs are denoted with hollow (p16-) and solid (p16+) white arrowheads. iCAFs are denoted with hollow orange arrowheads. (B) Quantification of percentage of p16+ cells in myCAF and iCAF subpopulations using human breast cancer tumor tissue arrays (n=51). (C) mIHC were performed on tumor DCIS samples using p16 (magenta), aSMA (green), PDGFRa (red), and pan-cytokeratin (PanCK; cyan) specific antibodies and nuclei were counterstained with hematoxylin and shown as dark blue. Cells triple positive for p16, aSMA, and PDGFRa are denoted by white arrowheads. Individual stains were pseudo colored as indicated and then merged (first panel). (D) Quantification of myCAFs (PDGFRa+ aSMA+ cells) in stroma (PanCK− cells) from multiplex IHC of DCIS samples that recurred (REC, n=7) or did not recur (No REC, n=9). (E) Quantification of p16+ myCAFs (PDGFRa+ aSMA+ cells) in stroma (PanCK− cells) from multiplex IHC of DCIS samples that recurred (REC, n=7) or did not recur (No REC, n=9). Unpaired one-tailed student t-test performed for all statistical analyses shown in this figure. All numerical data are represented as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001.

Figure 3.. Senescent CAFs are present in MMTV-PyMT murine mammary tumors.

(A) UMAP of stromal cells from 12-week-old MMTV-PyMT tumors (n=2). (B) Violin plots of CAF signature markers: myCAF—Pdgfra+, Acta2+, Thbs2+, Postn+; iCAF—Pdgfra+, Ly6c1+, Cxcl12+, Col14a1+; vCAF—Mcam+, Acta2+, Des+, Notch3+. (C) Dot plot displaying gene expression parameters including percentage of cells (size of black dots) and expression levels (colored scale bar) of a designated gene within a specific population (y-axis). Features include senescence markers (Cdkn2a (p16) and Cdkn2b (p15)) and SASP factors (Glb1, Col1a1, Col12a1, Mmp10, Mmp13, Cxcl9, Tnfsf11, Cd276, and Tgfb3). (D) Gene set enrichment analyses of the myCAF population compared to vCAF and iCAF showed in pathways including extracellular matrix organization, collagen assembly, and Fridman senescence. NES, p-value, and FDR are shown in each plot. (E) Feature plot and violin plot for the expression of Ncam1 in selected stromal cell types. (F) Correlation curve of Ncam1 and Cdkn2a (p16) expression in mammary stromal cell populations.

Figure 4.. NCAM1 marks senescent CAFs in MMTV-PyMT tumors.

(A) mIHC analyses were performed on 10-week-old MMTV-PyMT tumors using p16 (red), NCAM (green), PDGFRα (magenta), alpha-smooth muscle actin (aSMA) (cyan), Ly6C1 (orange), and MCAM (blue) specific antibodies and nuclei were counterstain with hematoxylin and shown as white (left: 1x; right: 25x). Individual stains were pseudo colored as indicated and then merged (first panel). myCAFs are denoted with hollow (p16-) and solid (p16+) white arrowheads. iCAFs were mostly p16- and are denoted with hollow orange arrowheads. (B) mIHC analyses were performed on 10 weeks old MMTV-PyMT tumors using p16 (red) and Ki67 (green) and nuclei were counterstained with hematoxylin and shown as dark blue (left: 5x; right: 25x). Greater than 70% of p16+ tumor cells were also positive for Ki67 and are denoted with hollow arrowheads. p16+ stromal cells were mostly negative for Ki67 and are denoted with solid arrowheads. (C) Quantification of percentage of Ki67+ cells out of p16+ tumor or stromal compartments as shown in B (n=4). (D) Four CAF populations were sorted from 10-week-old MMTV-PyMT mammary tumors (n=3) and subjected to bulk RNA-Seq analyses. Shown is heatmap highlighting CAF markers, senescence markers, and SASP factors. (E) Gene set enrichment analyses of the NCAM+ myCAF population compared to the remaining 3 CAF populations showed enrichment of the Fridman senescence signature. (F) Gene set enrichment analyses of the NCAM+ myCAF compared to the NCAM− myCAF population also showed enrichment of the Fridman senescence signature. NES, p-value, and FDR are shown in each plot. Unpaired one-tail student t-test was performed for C. All numerical data are represented as mean ± SEM. **** p<0.0001.

Figure 5.. Senescent CAFs drive MMTV-PyMT tumorigenesis.

(A) AP treatment regimen of MMTV-PyMT (INK-) and MMTV-PyMTxINKATTAC+ (INK+) mice. (B) Tumor volume measured with calipers weekly for INK− (n=16) and INK+ (n=19) mice. 2-way ANOVA was performed to compare the tumor growth; **p<0.01. (C) Representative IHC images for p16 expression in 7-week-old INK- and INK+ mice show predominant staining in stromal cells. (D) Quantification of p16 staining in tumor versus stromal compartment shown in (C) indicates that only stromal p16+ cells are lost in INK+ mice (n=5) compared to INK− mice (n=4). (E) Quantification of total myCAF population in tumors from 7 weeks old INK− versus INK+ mice. n=8 for each group. (F) Quantification of senCAF populations in tumors from 7 weeks old INK− versus INK+ mice. n=8 for each group. (G) Senolytic (ABT737) treatment regimen of MMTV-PyMT mice. (H) Tumor volume measured with calipers at week 7 for vehicle (Veh, n=8) and ABT737 (n=10) treated mice. (I) Quantification of total myCAF population in MMTV-PyMT mice treated with vehicle (Veh, n=9) or ABT737 (n=10). (J) Quantification of senCAF populations in MMTV-PyMT mice treated with Veh (n=9) or ABT737 (n=10). Unpaired one-tail student t-test was performed for all panels except for B. All numerical data are represented as mean ± SEM. *p<0.05; **p<0.01; ns, not significant.

Figure 6.. Senescent CAFs alter NK cells activation status and infiltration to increase tumor formation.

(A) Violin plot of indicated gene expression in different immune populations from 7-week-old AP treated MMTV-PyMT INK- and INK+ mice. (B) Violin plot of indicated gene expression in different immune populations from 7-week-old MMTV-PyMT mice treated with Veh or ABT737. (C) Flow cytometric quantification of intra-tumoral CD27-CD11b-hi NK cells in INK− (n=7) and INK+ (n=9) mice treated with AP. (D) Flow cytometric quantification of intra-tumoral CD27-CD11b-hi NK cells in MMTV-PyMT mice treated with Veh (n=5) or ABT737(n=6). (E) Representative IHC image for NK cells (NK1.1, red) and p16 (brown) in tumor sections from 7-week-old INK− (n=7) and INK+ (n=6) mice treated with AP. (F) Quantification of NK cells per tumor area from same mice shown in E. (G) Representative IHC image for NK cells (NK1.1, red) and p16 (brown) in tumor sections from 7-week-old MMTV-PyMT mice treated with Vehicle (Veh, n=8) or ABT737 (n=8). (H) Quantification of NK cells per tumor area from same mice shown in G. (I) NK cell depletion strategy in MMTV-PyMT (INK-) and MMTV-PyMTxINKATTAC+ (INK+) mice treated with AP. First dose of anti-NK1.1 or IgG isotype control antibody was administrated at 500 ug/mouse. The remaining doses were administrated every 4 days at 250 ug/mouse. (J) Tumor volume measured with calipers at week 7 for INK− versus INK+ mice depleted of NK cells (anti-NK1.1) or treated with control IgG, n=8. Unpaired one-tailed student t-test was performed for all statistical analyses shown in this figure. All numerical data are represented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ns, not significant.

Figure 7.. Senescent CAF-derived extracellular matrix limits NK tumor cell killing.

(A) Representative IHC image for NK cells (NK1.1, red) and p16 (brown) in tumor sections from 7 weeks old PyMT mice at 10X (left) and 40X (right) shows a close association between senCAFs and NK cells. (B) Gene set enrichment analyses of the senCAF population compared to the myCAF population showed enrichment of the collagen formation signature. NES, p-value, and FDR are shown in the plot. (C) Left: Representative image of Collagen (blue) and p16+ stromal cells (red). Collagen fibers imaged via second harmonic generation microscopy and analyzed via CT-FIRE/Curve align software. Fibers within 8μm of red p16+ stroma defined as p16+ (red arrowheads) and fibers at least 60μm away from p16+ stroma defined as p16- (green arrowheads). Right: Quantification of collagen fibers near p16- and p16+ stroma. Statistical difference determined via student t-test (n=3 mice, 3 FOV/mouse, unpaired student t-test, data are represented as mean ± SEM., ****p< 0.0001). (D) Cell chat analyses of collagen dialog between the different CAF and immune cell populations in tumors from 7-week-old PyMT mice. (E) Gene set enrichment analyses of the doxorubicin treated (i.e., senescent) compared to vehicle treated (i.e., control) fibroblasts also showed enrichment of collagen formation. NES, p-value, and FDR are shown. (F) 200,000 Py230 breast cancer cells labeled with luciferase were co-injected with 100,000 Ctrl or Sen fibroblasts and tumor growth was monitored by live animal bioluminescence imaging (BLI). Representative BLI of day 13 post tumor injection is shown. (G) Tumor growth was assessed by BLI on indicated time points (2-way ANOVA was performed to compare the tumor growth; data are represented as mean ± SEM. *p<0.05). (H) Quantification of CD3− NK1.1+ cells in Py230 tumors co-implanted with Ctrl (n=6) or Sen (n=6) fibroblasts on day 10 post tumor injection. (I) Quantification of CD27− CD11b-hi NK cells in Py230 tumors co-implanted with Ctrl (n=6) or Sen (n=6) fibroblasts on day 10 post tumor injection. (J) Tumor volume quantified with BLI for mice receiving Py230 tumor cells co-injected with Ctrl versus Sen fibroblasts under NK cell depletion treatment (day 13 post tumor inoculation). Py230-Ctrl fibroblasts group on IgG treatment, n=6; Py230-Sen fibroblasts group on IgG treatment, n=7; Py230-Ctrl fibroblasts group on anti-NK1.1 treatment, n=6; Py230-Sen fibroblasts group on anti-NK1.1 treatment, n=5. (K) Schematic of collagen deposition protocol. (L) Representative immunofluorescence of collagen 1A1 following the procedure outlined in K. (M) NK cell killing of tumor cells when plated on ECMs from Ctrl (n=6) or Sen (n=6) fibroblasts. Unpaired one-tailed student t-test was performed for all statistical analyses shown in this figure unless otherwise specified. All numerical data are represented as mean ± SEM. *p<0.05; **p<0.01; ns, not significant.