Search

Supplementary Figure Legend from Loss of Host Type-I IFN Signaling Accelerates Metastasis and Impairs NK-cell Antitumor Function in Multiple Models of Breast Cancer

Primer sequences used in realtime quantitative PCR

Figure legends for supplementary figures 1, 2 & 3

Genes altered between highly and weakly metastatic tumors

The effect of combination treatment on T cell activation

The lack of targeted therapies available for triple-negative breast cancer (TNBC) patients who fail to respond to first-line chemotherapy has sparked interest in immunotherapeutic approaches. However, trials utilizing checkpoint inhibitors targeting the PD-1/PD-L1 axis in TNBC have had underwhelming responses. Here, we investigated the interplay between type I IFN signaling and the PD-1/PD-L1 axis and tested the impact of combining IFN inducers, as immune activators, with anti–PD-1, to induce an antimetastatic immune response. Using models of TNBC, we demonstrated an interplay between type I IFN signaling and tumor cell PD-L1 expression that affected therapeutic response. The data revealed that the type I IFN-inducer poly(I:C) was an effective immune activator and antimetastatic agent, functioning better than anti–PD-1, which was ineffective as a single agent. Poly(I:C) treatment induced PD-L1 expression on TNBC cells, and combined poly(I:C) and anti–PD-1 treatment prolonged metastasis-free survival in a neoadjuvant setting via the induction of a tumor-specific T-cell response. Use of this combination in a late treatment setting did not impact metastasis-free survival, indicating that timing was critical for immunotherapeutic benefit. Together, these data demonstrated anti–PD-1 as an ineffective single agent in preclinical models of TNBC. However, type I IFN inducers were effective immune activators, and neoadjuvant trials combining them with anti–PD-1 to induce a sustained antitumor immune response are warranted. Cancer Immunol Res; 5(10); 871–84. ©2017 AACR.

IL-2 stimulated NK cells derived from Ifnar1-/- or WT mice have comparable cytotoxicity against syngeneic breast tumor lines.

A clinically relevant model of spontaneous breast cancer metastasis to multiple sites, including bone, was characterized and used to identify genes involved in metastatic progression. The metastatic potential of several genetically related tumor lines was assayed using a novel real-time quantitative RT-PCR assay of tumor burden. Based on this assay, the tumor lines were categorized as nonmetastatic (67NR), weakly metastatic to lymph node (168FARN) or lung (66cl4), or highly metastatic to lymph node, lung, and bone (4T1.2 and 4T1.13). In vitro assays that mimic stages of metastasis showed that highly metastatic tumors lines were more adhesive, invasive, and migratory than the less metastatic lines. To identify metastasis-related genes in this model, each metastatic tumor was array profiled against the nonmetastatic 67NR using 15,000 mouse cDNA arrays. A significant proportion of genes relating to the extracellular matrix had elevated expression in highly metastatic tumors. The role of one of these genes, POEM, was further investigated in the model. In situ hybridization showed that POEM expression was specific to the tumor epithelium of highly metastatic tumors. Decreased POEM expression in 4T1.2 tumors significantly inhibited spontaneous metastasis to the lung, bone, and kidney. Taken together, our data support a role for the extracellular matrix in metastatic progression and describe, for the first time, a role for POEM in this process.

Immune suppressor cell and T lymphocyte accumulation in the peripheral blood of WT and Ifnar1-/- mice.

Assays that do not distinguish metastatic phenotype

Metastatic progression is the major cause of breast cancer–related mortality. By examining multiple syngeneic preclinical breast cancer models in mice lacking a functional type-I interferon receptor (Ifnar1−/− mice), we show that host-derived type-I interferon (IFN) signaling is a critical determinant of metastatic spread that is independent of primary tumor growth. In particular, we show that bone metastasis can be accelerated in Balb/c Ifnar1−/− mice bearing either 4T1 or 66cl4 orthotopic tumors and, for the first time, present data showing the development of bone metastasis in the C57Bl/6 spontaneous MMTV-PyMT–driven model of tumorigenesis. Further exploration of these results revealed that endogenous type-I IFN signaling to the host hematopoietic system is a key determinant of metastasis-free survival and critical to the responsiveness of the circulating natural killer (NK)–cell population. We find that in vivo–stimulated NK cells derived from wild-type, but not Ifnar1−/−, mice can eliminate the 4T1 and 66cl4 breast tumor lines with varying kinetics in vitro. Together, this study indicates that the dysregulated immunity resulting from a loss of host type-I IFN signaling is sufficient to drive metastasis, and provides a rationale for targeting the endogenous type-I IFN pathway as an antimetastatic strategy. Cancer Immunol Res; 3(11); 1207–17. ©2015 AACR.

Anti-proliferative effects and production of type-I IFN by balb/c syngeneic breast tumor cell lines.

Loss of hematopoietic Ifnar1 reduces metastasis-free survival in the 4T1.2 model.

Metastasis is the major cause of death in patients with cancer; with no therapeutic cure, treatments remain largely palliative. As such, new targets and therapeutic strategies are urgently required. Here, we show that bone morphogenetic protein-4 (BMP4) blocks metastasis in animal models of breast cancer and predicts improved survival in patients. In preclinical models of spontaneous metastasis, BMP4 acted as an autocrine mediator to modulate a range of known metastasis-regulating genes, including Smad7, via activation of canonical BMP-SMAD signaling. Restored BMP4 expression or therapeutically administered BMP4 protein, blocked metastasis and increased survival by sensitizing cancer cells to anoikis, thereby reducing the number of circulating tumor cells. Gene silencing of Bmp4 or its downstream mediator Smad7, reversed this phenotype. Administration of recombinant BMP4 markedly reduced spontaneous metastasis to lung and bone. Elevated levels of BMP4 and SMAD7 were prognostic for improved recurrence-free survival and overall survival in patients with breast cancer, indicating the importance of canonical BMP4 signaling in the suppression of metastasis and highlighting new avenues for therapy against metastatic disease.Significance:Targeting the BMP4–SMAD7 signaling axis presents a novel therapeutic strategy to combat metastatic breast cancer, a disease that has had no reduction in patient mortality over 20 years.

PCR primers

C57BL/6 mice were injected intravenously with 10 μg (7.8E10 particles) of EO771 exosomes or liposomes every three days for 30 days. The number of cells per gram lung tissue of various immune cell populations in the lung was quantified by flow cytometry at endpoint: (A) CD45+ cells, (B) CD4 (CD3/CD4+) and CD8 T-cells (CD3+/CD8+), (C) NK cells (NK1.1+/CD3-), (D) dendritic cells (CD11C+/MHCII+), (E) macrophages (mø; CD11b+/F4/80+), and (F) CD11b+/Ly6Chigh monocytic MDSCs and CD11b+/Ly6Cmed granulocytic MDSCs. (G) Ratio of CD8+ T-cells to gMDSCs (CD11b+/Ly6Cmed) in the lung. Results are presented as mean {plus minus} s.e.m. (n=8-9 animals/group) and analyzed by 2-tailed Mann-Whitney U-tests, and Holm-Sidak multiple testing correction method; *: p

Legends for the four supplementary figures.

(A) Percentage of CD8, and CD4 T-cell death after exposure to EO771 exosomes as assessed by staining with the 7-aminoactinomycin D (7-AAD) viability dye. 1 μg of EO771 exosomes is equivalent to 7.9E9 particles. Representative histogram (flow cytometry profile) (left) and percentage of divided (right) (B) CD8+/CFSE+ T-cells, and (C) CD4+/CFSE+ T-cells following liposome treatment compared to PBS controls. The number of liposome particles is equivalent to 2 µg of EO771 exosomes (1.6E10), which was shown to significantly decrease T-cell proliferation. Results are presented as mean {plus minus} s.e.m. and analyzed by 2-tailed Mann-Whitney U-tests, and Holm-Sidak multiple testing correction method ***: p

BALB/C mice received a single intravenous injection of DiD-labeled 4T1- or 67NR-derived exosomes (20 µg; 1.2E11 particles). Liposomes injected at equivalent particle numbers served as control. Assessment of exosome uptake by immune cell lineages in the bone marrow (BM), spleen and lung was carried out 24-hours later by flow cytometry. (A) Representative flow cytometric plots (left) and quantification (right) of distinct DiD+ population within CD45+ cells. (B-G) Frequency of DiD+ subpopulation within (B) myeloid cells (CD11b+), (C) CD11b+/Gr1+ MDSCs, (D) dendritic cells (CD11C+/MHCII+), (E) macrophages (mø; CD11b+/F4/80+), (F) CD8 T-cells (CD3+/CD8+) and (G) CD4 T-cells (CD3+/CD4+). Results are presented as mean {plus minus} s.e.m. (n=5-7/group) as analyzed by ANOVA; *: p

C57BL/6 mice received a single intravenous injection of EO771-derived exosomes (50 µg/mouse; 4E11 particles) or liposomes. Immune composition changes in the lung was assessed 24-hours later by flow cytometry. Frequency of (A) myeloid cells (CD11b+), (B) dendritic cells (DC; CD11C+/MHCII+), (C) macrophages (mø; CD11b+/F4/80+), (D) Natural Killer cells (NK; NK1.1+/CD3-), (E) CD4 T-cells (CD3+/CD8+), and (F) CD8 T-cells (CD3+/CD8+). Results are presented as mean {plus minus} s.e.m. (n=5 animals/group) and analyzed by 2-tailed Mann-Whitney U-tests, and Holm-Sidak multiple testing correction method; *: p

Small membranous secretions from tumor cells, termed exosomes, contribute significantly to intercellular communication and subsequent reprogramming of the tumor microenvironment. Here, we use optical imaging to determine that exogenously administered fluorescently labeled exosomes derived from highly metastatic murine breast cancer cells distributed predominantly to the lung of syngeneic mice, a frequent site of breast cancer metastasis. At the sites of accumulation, exosomes were taken up by CD45+ bone marrow–derived cells. Subsequent long-term conditioning of naïve mice with exosomes from highly metastatic breast cancer cells revealed the accumulation of myeloid-derived suppressor cells in the lung and liver. This favorable immune suppressive microenvironment was capable of promoting metastatic colonization in the lung and liver, an effect not observed from exosomes derived from nonmetastatic cells and liposome control vesicles. Furthermore, we determined that breast cancer exosomes directly suppressed T-cell proliferation and inhibited NK cell cytotoxicity, and hence likely suppressed the anticancer immune response in premetastatic organs. Together, our findings provide novel insight into the tissue-specific outcomes of breast cancer–derived exosome accumulation and their contribution to immune suppression and promotion of metastases. Cancer Res; 76(23); 6816–27. ©2016 AACR.

Supplementary Figure Legends

Figure S3 shows that BMP4 does not alter migration or invasion in vitro and that BMP7 can also inhibit metastasis.

C57BL/6 mice were injected intravenously with 10 μg (7.8E10 particles) of EO771 exosomes or liposomes every three days for 30 days. Immune composition changes in the spleen was assessed at endpoint by flow cytometry. Frequency of (A) CD4 (CD3/CD4+) and CD8 T-cells (CD3+/CD8+), (B) NK cells (NK1.1+/CD3-), (C) CD11b+/Ly6Chigh monocytic MDSCs and CD11b+/Ly6Cmed granulocytic MDSCs, (D) dendritic cells (CD11C+/MHCII+) and (E) macrophages (mø; CD11b+/F4/80+). Results are presented as mean {plus minus} s.e.m. (n=9-10 animals/group) and analyzed by 2-tailed Mann-Whitney U-tests, and Holm-Sidak multiple testing correction method; *: p

Representative whole body fluorescent images of C57BL/6 mice at 4, 24 and 48-hours post-intravenous injection of Vybrant® DiD-labeled EO771 exosomes (20 µg; 1.6E11 particles) or liposomes at equivalent particle numbers using the IVIS® Spectrum (PerkinElmer) (n=5/group).

(A) Morphological characterization of exosomes isolated from the conditioned media of EO771 and 4T1 breast cancer cells by transmission electron microscopy. The scale bar indicates 200 nm. B) Detection of exosome-markers, CD9 (25 kDa), TSG101 (47 kDa), HSP70 (70 kDa) and cis-Golgi marker, GM130 (112 kDa) proteins in breast cancer exosomes (E) and cell lysates (C) by Western blotting. (C) Representative example of size distrbution of breast cancer exosomes as measured by Tunable Resistive Pulse Sensing (TRSPS; Izon qNano). (D) Quantification of the number of exosomes produced by EO771 and 4T1 breast cancer cells per milliliter of conditioned media under same culturing conditions. (E) The number of exosomes released from 1E6 67NR and 4T1 cells as quantified using TRSPS. Results are represented as mean {plus minus} s.e.m.

C57BL/6 mice received a single intravenous injection of EO771-derived exosomes (50 µg/mouse; 4E11 particles), or liposomes. Immune composition changes in the bone marrow was assessed 24-hours later by flow cytometry. Frequency of (A) myeloid cells (CD11b+), (B) dendritic cells (DC; CD11C+/MHCII+), (C) macrophages (mø; CD11b+/F4/80+), (D) Natural Killer cells (NK; NK1.1+/CD3-), (E) CD4 T-cells (CD3+/CD8+), and (F) CD8 T-cells (CD3+/CD8+). Results are presented as mean {plus minus} s.e.m. (n=5 animals/group) and analyzed by 2-tailed Mann-Whitney nonparametric U-tests, and Holm-Sidak multiple testing correction method.

Supplementary methods

AVI file - 4.5MB, Degradation of collagen IV by 4T1.2 cells + CA-074 inhibitor

PDF file - 296K, A)Regulation of Hif-1� and Hif-2alpha by hypoxia in breast cancer cells. B) Schematic of injection schedule

AVI file - 4.8MB, Degradation of collagen I by 4T1.2 cells

PDF file - 89K

PDF file - 293K

PDF file - 221K, A) Pre-metastatic niche induced by hypoxia increases melanoma metastasis. B) Factors identified in HCM of breast cancer cells

PDF file - 2.5MB

PDF file - 49K, A) Analysis of other immune cell population in the pre-metastatic niche. B) CD69 expression on NK cells in pre-metastatic niche. C) Confirmation of NK cell ablation with anti-asialoGM1

PDF file - 1.7MB

PDF file - 5.6MB

The TGFβ growth factor family member BMP4 is a potent suppressor of breast cancer metastasis. In the mouse, the development of highly metastatic mammary tumors is associated with an accumulation of myeloid-derived suppressor cells (MDSC), the numbers of which are reduced by exogenous BMP4 expression. MDSCs are undetectable in naïve mice but can be induced by treatment with granulocyte colony-stimulating factor (G-CSF/Csf3) or by secretion of G-CSF from the tumor. Both tumor-induced and G-CSF–induced MDSCs effectively suppress T-cell activation and proliferation, leading to metastatic enhancement. BMP4 reduces the expression and secretion of G-CSF by inhibiting NF-κB (Nfkb1) activity in human and mouse tumor lines. Because MDSCs correlate with poor prognosis in patients with breast cancer, therapies based on activation of BMP4 signaling may offer a novel treatment strategy for breast cancer. Cancer Res; 74(18); 5091–102. ©2014 AACR.

PDF file - 33K, A/B) Reduction of CD11b+/Ly6Cmed but not CD11b+/Ly6Chigh cells in pre-metastatic niche induced by HCM after MCP-1 neutralization

PDF file - 118K

AVI file - 4.7MB, Degradation of collagen IV by 4T1.2 CTSBkd cells

AVI file - 4.5MB, Degradation of collagen IV by 4T1.2 cells