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. 2020 Dec 1;80(23):5317-5329.
doi: 10.1158/0008-5472.CAN-20-2116. Epub 2020 Oct 6.

Fibroblast-Derived IL33 Facilitates Breast Cancer Metastasis by Modifying the Immune Microenvironment and Driving Type 2 Immunity

Ophir Shani  1 , Tatiana Vorobyov  1 , Lea Monteran  1 , Dor Lavie  1 , Noam Cohen  1 , Yael Raz  1 , Galia Tsarfaty  2 , Camila Avivi  3 , Iris Barshack  3 , Neta Erez  4
Affiliations

Fibroblast-Derived IL33 Facilitates Breast Cancer Metastasis by Modifying the Immune Microenvironment and Driving Type 2 Immunity

Ophir Shani et al. Cancer Res. .

Abstract

Lungs are one of the main sites of breast cancer metastasis. The metastatic microenvironment is essential to facilitate growth of disseminated tumor cells. Cancer-associated fibroblasts (CAF) are prominent players in the microenvironment of breast cancer. However, their role in the formation of a permissive metastatic niche is unresolved. Here we show that IL33 is upregulated in metastases-associated fibroblasts in mouse models of spontaneous breast cancer metastasis and in patients with breast cancer with lung metastasis. Upregulation of IL33 instigated type 2 inflammation in the metastatic microenvironment and mediated recruitment of eosinophils, neutrophils, and inflammatory monocytes to lung metastases. Importantly, targeting of IL33 in vivo resulted in inhibition of lung metastasis and significant attenuation of immune cell recruitment and type 2 immunity. These findings demonstrate a key function of IL33 in facilitating lung metastatic relapse by modulating the immune microenvironment. Our study shows a novel interaction axis between CAF and immune cells and reveals the central role of CAF in establishing a hospitable inflammatory niche in lung metastasis. SIGNIFICANCE: This study elucidates a novel role for fibroblast-derived IL33 in facilitating breast cancer lung metastasis by modifying the immune microenvironment at the metastatic niche toward type 2 inflammation.

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Conflict of interest statement

Conflict of interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. IL-33 is upregulated in metastases-associated fibroblasts at the lung metastatic microenvironment.
(A) Heatmap of inflammatory genes that were enriched in an unbiased GO analysis (GO:0006954, FDR=0.00045). Analysis was performed on genes significantly upregulated in MAF vs. NLF (FC>1.5, adjusted P<0.05). (B) Expression of Il33 in NLF and MAF sorted from MMTV-PyMT;Col1a1-YFP transgenic mice (n=3) vs. normal FVB/n;Col1a1-YFP controls (n=4) and BALB/c;Col1a1-YFP mice bearing 4T1 metastases (n=4) vs. BALB/c;Col1a1-YFP controls (n=3). Data are presented as fold change ± SD. Welch’s t-test, *p<0.05. (C) qRT-PCR analysis of Il33 expression in FACS sorted lung cell populations. Cells were isolated from MMTV-PyMT;Col1a1-YFP mice (n=3) or BALB/c;Col1a1-YFP mice bearing 4T1 tumor cell metastases (n=3). Data presented as mean ± SD, normalized to control immune cells; one-way ANOVA followed by Tukey’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001. (D) qRT-PCR analysis of Il33 expression. White bars: FVB/n lung fibroblasts incubated with SFM (NLF, n=7) or with Met1 tumor cells CM (ALF- activated lung fibroblasts, n=6). Gray bars: BALB/c NLF incubated with SFM (n=7) or ALF (n=7) incubated with 4T1 tumor cells CM. Data presented as mean ± SD; Welch’s t-test. **P<0.01, ****p<0.0001. (E) Scheme of IL-33 ELISA in lung homogenate supernatants. (F) Quantification of IL-33 ELISA presented in E: FVB/n normal lungs (n=7) vs. MMTV-PyMT metastases-bearing lungs (Mets, n=6) and BALB/c normal lungs (n=8) vs. BALB/c mice bearing 4T1 metastases (n=7). Data presented as mean ±SD; Welch’s t-test. *P<0.05. **P<0.01. (G) Scheme of IL-33 ELISA in BALF. (H) Quantification of IL-33 ELISA presented in G: FVB/n normal lungs (n=4) vs. MMTV-PyMT metastases-bearing lungs (n=10) and BALB/c normal lungs (n=4) vs. BALB/c mice bearing 4T1 metastases (n=3). Data presented as mean ± SD; Welch’s t-test. *P<0.05, **P<0.01. (I) Representative images of IL-33 immunofluorescence staining in lungs. n=3 mice per group; Scale bars, 50μm. Cell nuclei, DAPI; IL-33, Rhodamine Red. (J) Quantification of mean fluorescent intensity (MFI) per IL33+ positive cell in staining performed in (I). FVB/n normal lungs vs. PyMT metastases-bearing lungs, and BALB/c normal lungs vs. mice bearing 4T1 lung metastases. 6-8 fields of view - FOV/lung were analyzed. Mets adj: lung areas without metastases, Mets: metastases. One-way ANOVA with Tukey’s correction for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (K) Representative images of IL-33 and YFP immunofluorescence staining in lungs. n=3 mice per group; Scale bars, 50μm. Cell nuclei, DAPI; IL-33, Rhodamine Red; YFP, Alexa Fluor-488 (L) Representative CT imaging of 4T1 lung metastasis. Metastases are marked in red. n=4. (M) Pearson correlation analysis between metastatic volume (mm3) measured by CT imaging presented in (L) and Il33 expression analyzed by qRT-PCR in total lungs.
Figure 2
Figure 2. Lung metastases formation is characterized by extensive modifications in the immune milieu.
(A,B) qRT-PCR analysis of Il33 downstream genes (A) and immune related TFs (B) in total lungs derived from BALB/c mice bearing 4T1 tumor cell metastases (n=4) and in normal controls (n=5). Data are presented as mean ± SD of technical repeats; Multiple t-tests with Welch’s correction, FDR(Q)=5% and Fold Change>1.5. **q<0.01, ***q<0.001, ****q<0.0001. ns-not significant. (C) Heat map presenting Pearson correlation analysis between Il33 expression and the expression of the genes presented in (A,B). Results are presented as Pearson R values and P values per gene. Significant results were considered as P<0.05 and R>I0.651 (D) Flow cytometry analysis of the number of immune cells in normal lungs and metastases bearing lungs derived from FVB/n normal lungs (Normal, n=5) vs. MMTV-PyMT metastases-bearing lungs (Mets, n=5) and BALB/c normal lungs (n=5) vs. BALB/c mice bearing 4T1 tumor cell metastases following orthotopic injection (n=4). Data presented as mean ± SD. Welch’s t-test, **P<0.01. (E) Quantification of flow cytometry analysis of immune cell populations in the lung derived from FVB/n normal lungs (Normal, n=5) vs. MMTV-PyMT metastases-bearing lungs (Mets, n=5) and BALB/c normal lungs (n=5) vs. BALB/c mice bearing 4T1 tumor cell metastases following orthotopic injection (n=4). Data presented as mean ± SD of percent out of CD45+ cells. Gating strategy presented in Supplementary Fig. S2C,D. (F-J) Cell numbers of different populations based on quantification performed in (E), Monocytes presented in (F), Macrophages presented in (G), Lymphoid cells presented in (H), granulocytes presented in (I), Dendritic cells in (J), Two-way ANOVA with Sidak’s correction for multiple comparisons. **P<0.01, ***P<0.001 ****P<0.0001.
Figure 3
Figure 3. ST2, the IL-33 receptor is highly upregulated in immune cells at the metastatic microenvironment
(A) qRT-PCR analysis of St2 expression in FACS sorted lung cell populations: immune cells (CD45+CD31-), endothelial cells (CD31+CD45-), epithelial/tumor cells (EpCAM+CD45- CD31-) or fibroblasts (YFP+CD45-CD31-EpCAM-). Cells were sorted by flow cytometry from MMTV-PyMT;Col1a1-YFP mice (n=3), FVB/n;Col1a1-YFP (n=4) or BALB/c;Col1a1-YFP mice bearing 4T1 tumor cell metastases (n=4) or normal BALB/c;Col1a1-YFP control (n=5). Data presented as mean ±SD normalized to control immune cells; one-way analysis of variance followed by Tukey’s multiple comparisons test. **p<0.01, ***p< 0.001, **** p <0.0001. (B) Flow cytometric analysis of the number of ST2+CD45+ immune cells in FVB/n normal lungs (n=5) vs. MMTV-PyMT metastases-bearing lungs (n=5) and BALB/c normal lungs (n=7) vs. BALB/c mice bearing 4T1 tumor cell metastases following orthotopic injection (n=7). Data presented as mean ±SD; Welch’s t-test **P<0.01. (C) Flow cytometry representative histogram of ST2 fluorescent intensity (MFI) out of total CD45+ immune cells in normal lungs and metastases-bearing lungs (Mets). Histograms of Normal and Mets represent individual mice. FMO-fluorescent minus one. (D) Quantification of flow cytometric analysis presented in (C). Each sample was normalized to FMO control and to normal control. Welch’s t-test, *P<0.05, ****p<0.0001. (E-H) Flow cytometric analysis of ST2 fluorescence intensity in immune cell populations in normal lungs and metastases-bearing lungs derived from FVB/n normal lungs (n=3) vs. MMTV-PyMT metastases-bearing lungs (n=3) and from BALB/c mice injected with 4T1 tumor cells (n=4) or control BALB/c mice (n=4). Lymphoid cells (E), Monocytes or Macrophages (F), Granulocytes (G), Dendritic cells (H). Gating strategy presented in Supplementary Fig. S2C,D. Each cell population was normalized to FMO control and to normal control. Multiple t-tests with Welch’s correction, FDR(Q)=5%. *q<0.05, **q<0.01, ***q<0.001, ns-not significant.
Figure 4
Figure 4. IL-33 is functionally important for direct recruitment of T cells and eosinophils.
(A) Scheme for in vitro migration assays of neutrophil, monocyte and T cells. Bone marrow-derived neutrophils and monocytes were isolated by gradient separation. CD3+ T cells were purified from spleen. (B-D) Quantification of neutrophil (B) monocytes (C) and T cells (D) migration based on experimental design described in (a). One-way ANOVA with Tukey’s correction for multiple comparisons tests. *P<0.05, **P <0.01, ***P<0.001, ****P<0.0001. (E) Scheme for eosinophil isolation and in vitro maturation, followed by migration assay. Eosinophil purity analysis is presented in Supplementary Fig. S3. (F) Quantification of eosinophils migration. One-way ANOVA with Tukey’s correction for multiple comparisons tests. *P<0.05, **P <0.01.
Figure 5
Figure 5. Inhibition of IL-33 attenuates lung metastases, and tempers type-2 immunity in the metastatic lung.
(A) Experimental design of IL-33 inhibition in vivo. Experiments were performed 5 separate times with at least n=5 mice per group. (B) Representative CT scans of mice injected as described in (A). Metastases are circled in red. Scale bars: 3mm. (IC: isotype control, αIL33: Anti-IL33 Antibody). (C) Number of metastatic foci as identified by CT scans. Welch’s t-test, ****P<0.0001. (D) Area of metastases per mouse as measured by CT scans. Welch’s t-test, **P<0.01. (E) Representative H&E staining of lungs injected as described in (A), metastases are circled in red. Scale bars: 3mm (F) Quantification of H&E staining for the metastatic area out of total lung area (metastatic burden). n=19 lung tissue sections of 4 mice. Welch’s t-test, ****P<0.0001. (G) Quantification of H&E staining metastatic area per mouse. n=4 mice. Welch’s one-tailed t-test, *P<0.05. (H) Flow cytometry Immune cell populations analysis in mice injected as described in (A). Multiple t-tests with Welch’s correction, FDR(Q)= 1%. *q<0.05, **q<0.01, ****q<0.0001. (I,J) Expression analysis by qRT-PCR of selected genes in lungs derived from mice injected with α-IL-33 or with IC, as described in (a). n≥11 mice per group. Multiple t-tests with Welch’s correction, FDR(Q)=5%. *q<0.05, **q<0.01, ns-not significant.
Figure 6
Figure 6. IL-33 is upregulated in the stroma of breast cancer primary tumors and lung metastases in human patients.
(A) IL33 expression in tumor-associated stroma as compared with normal stroma from GSE9014. Welch’s t-test, *P<0.05. (B) IL33 expression in paired patient samples of epithelial cells and tumor-associated stroma in primary breast tumors from GSE88715. Paired t-test. **P<0.01. (C) IL33 expression in primary breast-tumor tissue (Epi) and tumor associated-stroma (Stroma) from GSE14548. Stromal expression is divided into tumor grade (Stroma-grade 1: G1; Stroma-grade 2: G2; Stroma-grade 3: G3). For the epithelial expression of IL33 all grades were combined (G1-3). One-way ANOVA with Tukey’s correction for multiple comparisons, *P<0.05, **P<0.01, ****P<0.0001. (D) IL33 mRNA expression in human breast cancer metastasis from different metastatic sites. Data was derived from GSE14020. One-way ANOVA with Tukey’s correction for multiple comparisons tests. *P<0.05, **** P< 0.0001. (E) Representative IHC staining of IL-33 in lung metastases of breast cancer patients (n=8). Normal areas (remote from metastatic foci) were quantified as controls. Scale bars: 200μm. (F) Quantification of the number of IL-33+ cells per FOV in IHC performed in (c). 10-15 FOV/section. Data presented as mean ±SD; Welch’s t-test. ****P<0.0001. (G) Analysis of the mean IL-33+ cells/patient, compared with paired adjacent normal lung tissue. n=8; Paired t-test. *P<0.05.
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