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Granulocytic immune infiltrates are essential for the efficient formation of breast cancer liver metastases
Sébastien Tabariès1,2,
Véronique Ouellet4,
Brian E Hsu1,2,
Matthew G Annis1,2,
April AN Rose1,2,
Liliane Meunier4,
Euridice Carmona4,
Christine E Tam1,2,
Anne-Marie Mes-Masson4,5 &
Peter M Siegel1,2,3
Breast Cancer Research volume 17, Article number: 45 (2015) Cite this article
Breast cancer cells display preferences for specific metastatic sites including the bone, lung and liver. Metastasis is a complex process that relies, in part, on interactions between disseminated cancer cells and resident/infiltrating stromal cells that constitute the metastatic microenvironment. Distinct immune infiltrates can either impair the metastatic process or conversely, assist in the seeding, colonization and growth of disseminated cancer cells.
Using in vivo selection approaches, we previously isolated 4T1-derived breast cancer cells that preferentially metastasize to these organs and tissues. In this study, we examined whether the propensity of breast cancer cells to metastasize to the lung, liver or bone is associated with and dependent on distinct patterns of immune cell infiltration. Immunohistocytochemistry and immunohistofluorescence approaches were used to quantify innate immune cell infiltrates within distinct metastases and depletion of Gr1+ (Ly-6C and Ly-6G) or specifically Ly-6G+ cells was performed to functionally interrogate the role of Ly-6G+ infiltrates in promoting metastasis to these organs.
We show that T lymphocytes (CD3+), myeloid-derived (Gr-1+) cells and neutrophils (Ly-6G+ or NE+) exhibit the most pronounced recruitment in lung and liver metastases, with markedly less recruitment within bone metastatic lesions. Interestingly, these infiltrating cell populations display different patterns of localization within soft tissue metastases. T lymphocytes and granulocytic immune infiltrates are localized around the periphery of liver metastases whereas they were dispersed throughout the lung metastases. Furthermore, Gr-1+ cell-depletion studies demonstrate that infiltrating myeloid-derived cells are essential for the formation of breast cancer liver metastases but dispensable for metastasis to the lung and bone. A specific role for the granulocytic component of the innate immune infiltrate was revealed through Ly-6G+ cell-depletion experiments, which resulted in significantly impaired formation of liver metastases. Finally, we demonstrate that the CD11b+/Ly-6G+ neutrophils that infiltrate and surround the liver metastases are polarized toward an N2 phenotype, which have previously been shown to enhance tumor growth and metastasis.
Our results demonstrate that the liver-metastatic potential of breast cancer cells is heavily reliant on interactions with infiltrating Ly-6G+ cells within the liver microenvironment.
Communication between the tumor and surrounding stromal cells is a critical determinant governing the ability of cancer cells to metastasize to specific organs. The tumor microenvironment consists not only of extracellular matrix proteins, resident fibroblasts and endothelial cells, but also infiltrating innate (macrophages, neutrophils, myeloid-derived suppressor cells or natural killer cells) and adaptive (B and T lymphocytes) immune cells [1]. Leukocyte infiltrates are present in the majority of solid tumors; however, the functional roles and clinical consequences of these immune cell infiltrates are complex [2]. In some circumstances, the ability of inflammatory cells to destroy tumor cells has been associated with better prognosis [3,4]. In contrast, numerous studies have shown that inflammation can also contribute to the establishment of primary tumors and subsequent metastasis by allowing tumor cells to escape and/or actively suppress anti-tumor immune responses [2,5,6]. While cancer-related inflammation has mostly been studied in the context of primary tumor growth, it is now accepted that inflammatory cells and secreted mediators are also involved in the migration, invasion and metastasis of malignant cells [2].
Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) are well-characterized infiltrating innate cell populations that augment breast cancer metastasis [7], in part, through their ability to stimulate tumor angiogenesis and suppress anti-tumor immunity [8,9]. Infiltration of MDSCs, which are defined as CD11b/Gr-1 double-positive myeloid cells, into the primary tumor and metastatic sites is often associated with poor prognosis in breast cancer patients [10,11]. MDSCs suppress both innate and adaptive immune responses resulting in diminished effector T cell functions [6,12]. In addition, MDSCs also promote the activation and expansion of regulatory T cells to mediate immunosuppression [13]. MDSC accumulation at distant metastatic sites, which contributes to the establishment of a pre-metastatic niche, has also been reported to enhance metastatic efficiency [14]. Thus, the pre-metastatic niche may provide 'privileged sites' for tumor cells to adhere and successfully colonize different organs and tissues [15,16].
Neutrophils are garnering attention as important modulators of cancer progression [17-20]. Like TAMs, tumor-associated neutrophils (TANs) may exist in different states of activation/differentiation [21]. TANs can adopt either an anti-tumorigenic (N1) or a pro-tumorigenic (N2) phenotype. Thus, N1-polarized neutrophils have the potential to kill cancer cells and inhibit tumor growth [22-24] as well as coordinate adaptive immune responses through interactions with dendritic cells [25]. In contrast, N2 neutrophils may support tumor growth by producing pro-angiogenic factors and matrix-degrading enzymes, suppress anti-tumor immune responses and facilitate metastasis [21,26-28]. Thus, infiltrating innate immune cell populations, such as TAMs, MDSCs or TANs, can play complex roles during cancer initiation and progression to metastasis. To understand how these cell populations influence general and/or organ-specific breast cancer metastasis, we employed 4T1 breast cancer cell populations isolated from primary mammary tumors, as well as those explanted from bone, lung or liver metastases. Our data reveal that breast cancer cells derived from different metastatic sites are characterized by unique and overlapping patterns of chemokine expression and secretion. Interestingly, liver-metastatic breast cancer cells display high levels of chemokines that recruit a variety of innate immune cell populations. Using antibody-mediated depletion of Ly-6G+ cells in immune-competent mice, we uncover a pro-metastatic function for this specific component of the innate immune infiltrate, which is required for the establishment of liver metastases but dispensable for bone and lung metastases.
Cell culture and transfections
The 4T1 cell line was obtained from the American Type Culture Collection and cultured as previously described [29]. The isolation of 4T1-derived cells explanted from primary tumors (148, 152 or 154), 4T1-derived liver-aggressive cell populations (2776, 2792 and 2869), 4T1-derived lung-aggressive cell populations (526, 533 and 537) and 4T1-derived bone-aggressive cell populations (590, 592 and 593) were described previously [29-31].
RNA amplification, labeling and hybridization to Agilent microarrays
RNA was extracted from 4T1 parental and individual in vivo selected metastatic subpopulations, purified total RNA was subjected to amplification and hybridized to 44 K whole mouse genome microarray gene expression chips (Agilent Technologies, Santa Clara, CA, USA) as described previously [30].
Microarray expression data were normalized and analyzed using GeneSpring software (V7.3, Agilent Technologies). Each group (parental cell line and the explants derived from mammary fat pad, bone, lung and liver metastasis) was individually compared to all the other groups. A two-fold change cutoff was determined and differentially expressed genes were selected using a nonparametric test coupled with a false discovery rate of 0.05. The microarray data can be accessed through the GEO repository (ID GSE62598).
Bioinformatics analysis
Analysis of differentially expressed genes was performed in the context of gene ontology, canonical pathways and molecular networks using the Ingenuity Pathways Analysis (IPA, Ingenuity Systems™, Qiagen, Redwood City, CA, USA). The differentially expressed genes were compared to genetic categories in the IPA database, and ranked according to P values.
Chemokine array
To detect and quantify chemokines secreted by each cell population, serum-free conditioned media (CM) was collected from cells cultured for 48 h and applied to a chemokine (mouse) quantitative antibody array (Abnova Corp., Taipai, Taiwan, cat#AA0118) following the manufacturer's protocol. Array scans were acquired with a GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA, USA) at a 5 uM/pixel resolution and features were extracted with the GenePix 6.1 software. The experiment was performed in duplicate.
Immunohistocytochemistry
For CD3 staining, tissue sections were stained with the Benchmark XT autostainer (Ventana Medical Systems, Inc., Tucson, AZ, USA). Antigen retrieval was performed using Cell Conditioning 1 (Ventana Medical Systems, Inc., cat#950-124) for 60 minutes, following the manufacturer's instructions. Pre-diluted antibody (CD3: 1:100, Abcam, Cambridge, MA, USA, cat#ab16669) was manually added to the slides and incubated at 37°C for 60 minutes. Reactions were performed using the iView DAB detection kit without secondary antibody (Ventana Medical Systems, Inc., cat#760-093), where pre-diluted anti-rabbit secondary antibody (1:300; Santa Cruz Biotechnology, Dallas, TX, USA) was automatically dispensed. Counterstaining was achieved with hematoxylin and bluing reagent (Ventana Medical Systems, Inc., cat#760-2021 and cat#760-2037).
For Ly-6G or neutrophil elastase (NE) staining, unstained sections (4 μm) were subjected to antigen retrieval in 10 mM citrate buffer (pH 6.0) for 20 minutes at sub-boiling temperatures. Slides were incubated overnight at 4°C with a rabbit anti-Ly-6G antibody (clone 1A8; 1:200 dilution, BD Pharmingen, San Jose, CA, USA, cat#551459) or with a rabbit anti-neutrophil elastase antibody (1:200 dilution, Abcam, cat#ab68672). Following incubation with the primary antibody, secondary biotin-conjugated antibodies were applied for 45 minutes. After washing with phosphate-buffered saline (PBS), slides were developed with diaminobenzidine (Dako, Glostrup, Denmark) as the chromogen and counterstained with Harris hematoxylin. All slides were scanned using a Scanscope XT digital slide scanner (Aperio, Vista, CA, USA). Scans were quantified by analyzing positive staining with Imagescope software (Aperio) using the positive pixel count algorithm. In all analyses, only moderate (+2) and strong (+3) staining was considered as positive pixels. For all staining, data are expressed as a ratio of positive pixels over the total pixels per field.
Unstained sections (4 μm) were blocked in 1% bovine serum albumin (BSA) with 2% horse serum and 0.05% Tween-20 for 30 minutes. Slides were incubated overnight at 4°C with a goat anti-S100a8 antibody (1:50 dilution, Santa Cruz Biotechnology, cat#sc-8112) and with a rabbit anti-NE antibody (1:50 dilution, Abcam, cat#ab68672). Following incubation with the primary antibody, sections were incubated with Alexa Fluor 555-conjugated donkey anti-goat (cat#A-21432; Molecular Probes, Eugene, OR, USA) and Alexa Fluor 647-conjugated donkey anti-rabbit (Molecular Probes, cat#A-31573) secondary antibodies. Nuclei were counterstained with 4′, 6′-diamidino-2-phenylindole (DAPI), and images were taken (63X) using a Zeiss LSM 710 confocal on an Axio Observer full-motorized inverted microscope (Carl Zeiss Microscopy, Jena, Germany). Images were analyzed with MetaXpress software (Molecular Devices, Sunnyvale, CA, USA). Data is expressed as a percentage of cells double positive for S100a8 and NE over the total number of S100a8-positive cells. Two independent reviewers scored the co-localization of these markers (BEH, CET).
Finally, OCT-embedded livers were sectioned (10 μm) and fixed in methanol for 10 minutes. The sections were blocked with 2% BSA and 1% normal horse serum (Vector Laboratories, Burlingame, CA, USA, cat#S-2000) and subsequently incubated overnight at 4°C with a primary antibody directed against Ly-6G (clone 1A8; 1:100 dilution, BD Pharmingen, cat#561236), CD11b (clone M1/70, 1:100 dilution, eBioscience, San Diego, CA, USA cat#53-0112) and MMP9 (1:100 dilution, Abcam, cat#ab38898). Following incubation with the indicated primary antibodies, Alexa Fluor 555-conjugated donkey anti-rabbit (Molecular Probes, cat#A-31572) secondary antibodies were applied to each section. Nuclei were counterstained with DAPI, and images were taken (63X) using a Zeiss LSM 710 confocal on an Axio Observer full-motorized inverted microscope (Carl Zeiss Microscopy). Images were analyzed with MetaXpress software (Molecular Devices) and the data expressed as a percentage of Ly-6G+CD11b+MMP9+ triple-positive cells (N2-polarized neutrophils) over the total number of Ly-6G+CD11b+ double-positive cells (neutrophils). Three independent reviewers scored the co-localization of these markers (VO, BEH and ST).
Experimental metastasis assays
Experimental liver metastasis assays (1 × 105 cells; splenic injection) and experimental lung metastasis assays (5 × 105 cells; tail vein injection) were performed as previously described [30,32]. Tumor burden in the liver (left cardiac lobe) and lungs was quantified as previously reported [30]. The tumor area/tissue area was quantified using Imagescope software (Aperio). Experimental bone metastasis assays (1 × 105 cells; left cardiac ventricle injection) as well as X-ray micro-computed tomography (μCT) imaging was conducted as previously described [33]. All mice were sacrificed two weeks following injection. The mice were housed in facilities managed by the McGill University Animal Resources Centre and all animal experiments were conducted under a McGill University-approved Animal Use Protocol (AUP #5129), which was reviewed by the Facility Animal Care Committee for the Faculty of Medicine (Committee A). All projects involving live animals were subjected to peer review for scientific merit, in accordance with guidelines established by the Canadian Council on Animal Care.
Antibody-mediated depletion experiments
To deplete Gr-1+ cells in vivo, purified anti-Ly-6G/Ly-6C rat monoclonal antibody (mAb) (clone RB6-8C5, AbLab, University of British Columbia, BC, Canada) or control isotype immunoglobulin G (IgG) were administered to cohorts of mice. In all cases, mice were treated by intraperitoneal (i.p.) injection (1.5 μg/g) of purified antibodies. Mice were first treated 24 h prior to tumor cell injection and every 72 h thereafter. Differential leukocyte counts were performed at the completion of each experiment and revealed greater than 90% depletion of peripheral blood neutrophils using these treatment regimes.
To specifically deplete Ly-6G+ cells in vivo, purified anti-Ly-6G rat mAb (clone 1A8, BioXcell, West Lebanon, NH, USA) or control isotype IgG (clone 2A3, BioXcell) were administered to cohorts of mice. Mice were treated by i.p. injection (5.5 μg/g) of purified antibodies as indicated. Briefly, mice were first treated 24 h prior to tumor cell injection and every 48 h thereafter. Differential leukocyte counts were performed at the completion of the experiment and revealed greater than 94% depletion of peripheral blood neutrophils using these treatment regimes. At the experimental endpoint, immunohistochemical staining for Ly-6G revealed up to 65% depletion of liver-infiltrating neutrophils in mice treated with the 1A8 antibody versus the 2A3 isotype control. Quantification of Ly-6G staining was performed as described above (Immunohistocytochemistry).
CD11b+/Ly-6G+ cell isolation
Seven days or 14 days following splenic injection of the breast cancer cells, CD11b+/Ly-6G+ cells were isolated from the metastasis-bearing livers. Livers were first perfused with perfusion medium (Gibco, Waltham, MA, USA, cat#17701-038) for 10 minutes at 5 ml/min. Following perfusion, livers were minced and dissociated for 1 h in a mix of collagenase A, collagenase B and hyaluronidase (2 mg/ml each, Roche, Basel, Switzerland, cat#11088793001 and 11088031001; Sigma-Aldrich, St Louis, MO, USA, cat#H3884).
To purify CD11b+/Ly-6G+ cells [34,35], cells were blocked in 2.4G2 (1:10 dilution) for 30 minutes and then incubated for 30 minutes, in the dark, with an antibody cocktail containing anti-mouse CD11b-APC (1:10000 dilution, eBioscience, clone M1/70, cat#17-0112-82) and rat anti-mouse Ly-6G-PerCP-Cy5.5 (1:1000 dilution, BD Biosciences, San Jose, CA, USA, clone 1A8, cat#560602). Subsequently, cells were washed once with PBS and then incubated with Live/Dead stain (1:1000 dilution) for 30 minutes. Cells were then washed once with PBS and resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS complemented with 2% fetal bovine serum (FBS)). Samples were read on the BD FACS Canto (BD Biosciences).
RNA isolation and real-time RT-PCR
Total RNA from isolated CD11b+/Ly-6G+ neutrophils (isolated as described above) was extracted and reverse transcribed as previously described [30]. Following the reverse transcription reaction, all samples were diluted 1:50 in ddH2O and subjected to real-time PCR analysis with FastStart Universal Probe Master (Roche, cat#04913914001). Ten picograms of gene-specific primers (IFN-β sense: 5′-TCCATCATGAACAACAGGTG-3′; IFN-β antisense: 5′-GACATTTCCGAA TGTTCGTC-3′; CCL3 sense: 5′-TGTACCATGACACTCTGCAAC-3′; CCL3 antisense: 5′-CA ACGATGAATTGGCGTGGAA-3′; TNF-α sense: 5′-TCGGGGTGATCGGTCCCCAA-3′; TNF-α antisense: 5′-GGTGGTTTGCTACGACGTGGGC-3′; MMP9 sense: 5′-TCGCGTGGAT AAGGAGTTCT-3′; MMP9 antisense: 5′-CGGTTGAAGCAAAGAAGGAG-3′; CCL5 sense: 5′-CCTCACCATATGGCTCGGACACC-3′; CCL5 antisense: 5′- GCGCGAGGGAGAGGTAG GCA-3′; Arg-1 sense: 5′-CTCCAAGCCAAAGTCCTTAGAG-3′; Arg-1 antisense: 5′- GGAGCTGTCATTAG GGACATCA-3′) were used in a total reaction volume of 15 μl. For all targets, the following cycling conditions were: 95°C for 10 minutes, followed by 40 cycles each consisting of 95°C for 15 seconds, 60°C for 30 seconds and 72°C for 45 seconds. Incorporation of SYBR Green dye into the PCR products was monitored using a Rotor Gene RG-3000 Real-time PCR system (Roche). Pfaffl analysis method was used to measure the relative quantity of gene expression [36]. The reference gene, Gapdh (Gapdh sense: 5′-CAAGTATGATGACATCAAGAAGGTGG-3′; Gapdh antisense: 5′-GGAAGAGTGGGAGTT GCTGTTG-3′) was selected based on its stable expression in all cell populations analyzed. Relative mRNA levels were expressed as relative expression comparing early CD11b+/Ly-6G+ neutrophils with late CD11b+/Ly-6G+ neutrophils. All measurements were done in duplicate from four independent sets of injections.
The significance values associated with the positivity for CD3, Ly-6G (clone 1A8), NE, co-localization of s100a8 and NE and the formation of bone, lung or liver metastases from breast cancer cells in mice depleted for Gr-1+ cells or Ly-6G+ cells versus isotype controls were calculated using a Student t test.
Gene expression profiling reveals distinct expression patterns associated with 4T1 subpopulations derived from different metastatic sites
We previously reported the isolation of 4T1-derived subpopulations explanted from primary breast tumors (148, 152 and 154) [31] as well as in vivo selected cells that grow aggressively in bone (590, 592 and 593) [29], lung (526, 533 and 537) [31] or liver (2776, 2792 and 2869) [30] (Figure 1A). To identify mediators that contribute to general and/or organ-specific metastatic abilities of breast cancer cells, we performed gene expression profiling using Agilent whole mouse genome microarrays on each of these cell populations (Figure 1B). This approach yielded 395 differentially expressed genes (fold change: 2; false discovery rate: 0.05; see Additional file 1), which, when used to hierarchically cluster the cell populations, could largely segregate them according to the site from which they were derived (Figure 1C).
Gene expression profiling reveals distinct expression patterns associated with different metastatic 4T1 subpopulations. (A) Schematic depicting 4T1-derived breast cancer cell populations isolated from distinct metastatic sites. (B) To identify candidate genes that were differentially expressed among the isolated populations, each group (parental cell line and the explants derived from mammary fat pad (PE), bone, lung and liver metastases) was individually compared to all the other groups. A two-fold change cutoff was determined and 395 differentially expressed genes were selected using a nonparametric test coupled with a 5% false discovery rate. (C) A heatmap displaying the hierarchical clustering of isolated 4T1-derived cell populations using the 395 differentially expressed genes. Red color indicates those genes that are highly expressed and the green color denotes those genes that are underexpressed. The majority of cell populations clustered according to the site from which they were derived.
4T1-derived metastatic breast cancer populations possess distinct chemokine profiles
We next subjected the 395 genes to Ingenuity Pathway Analysis (IPA), which revealed 34 significantly affected pathways (Table 1). Of these, a granulocyte adhesion and diapedesis-associated gene signature was the most prominent pathway affected in the various 4T1-derived explants. Intriguingly, other pathways implicating immune cell responses such as IL8 signaling, antigen presentation or leukocyte extravasation were also identified (Table 1). These pathways are characterized by the differential expression of several common genes, including those encoding chemokines (Table 1), which are known to induce directed chemotaxis in responsive leukocyte populations. To determine the repertoire of chemokines secreted by the 4T1-derived breast cancer populations, we employed a multiplex chemokine antibody array (Figure 2). Using this approach, we found that CCL2 and CXCL2, which are chemotactic for inflammatory monocytes and granulocytes [37,38], respectively, were secreted at higher levels by all metastasis-derived cell populations compared to primary tumor explants and the parental cell line (Figure 2). The bone- and lung-aggressive populations exhibited the highest degree of variability in expression of many chemokines; whereas the liver-metastatic populations were also characterized by uniformly high levels of CXCL9 and CCL9 (Figure 2), which are known to recruit T lymphocytes and neutrophils, respectively [39,40]. Collectively, this data demonstrate that cell populations, selected for their ability to metastasize to distant organs, are characterized by an increased production of chemokines and that the pattern of secreted chemokines differs depending on the metastatic site from which they were derived.
Table 1 Differentially affected pathways by Ingenuity Pathway Analysis
4T1-derived breast cancer populations possess distinct and overlapping chemokine profiles. A heatmap representing the secreted levels of 25 chemokines measured using a chemokine array. The chemokines are grouped based on the immune cell types that they are known to recruit (multiple immune cell types, macrophages and/or lymphocytes, T lymphocytes and/or neutrophils, macrophages, T lymphocytes or neutrophils). Red color indicates chemokines that are highly expressed and the blue color denotes chemokines that are underexpressed.
T lymphocytes are recruited to breast cancer liver and lung metastases
Metastatic breast cancer cells expressed chemokines that can direct the recruitment of T lymphocytes (CXCL9, CCL2 or CX3CL1) [40-42]. Immunohistocytochemistry staining with an antibody against a pan T cell marker (CD3) revealed positivity in all samples examined (Figure 3A,B). Indeed, CD3-positive staining was found enriched in both lung and liver metastases compared to bone metastases or primary tumors (Figure 3B). In addition, CD3-positivity was higher in lung or liver metastases compared to tissue adjacent (ADJ) to the metastases or control (CTRL) samples that were devoid of lesions (Figure 3B). However, while CD3-positive cells were found throughout the lung lesions, about 2/3 of liver lesions revealed increased abundance of CD3-positive cells at the periphery of the metastatic lesions (Figure 3). These data indicate that T lymphocyte recruitment is associated with lung and liver metastases and localization differs depending on metastatic site.
T lymphocytes are recruited to liver and lung metastases from breast cancer. Paraffin-embedded sections from primary breast tumors, bone specimens, lung specimens and liver specimens obtained following experimental metastasis assays were subjected to immunohistochemical staining with anti-CD3 antibodies. (A) Representative images from 20X, 40X magnifications for each site are shown. 40X images were taken at the margin of the lesions (40X margin) and in more distal regions (40X adj.). Scale bar represents 40 μm (20X) or 20 μm (40X) and applies to all panels of the same magnification. (B) Positivity of CD3 staining (expressed as a ratio of positive pixels over the total pixels per field) quantified inside lesions (TUMOR), in the adjacent tissue (ADJ) or in control (CTRL) samples without any lesions. Lymphocyte T expression and recruitment is mostly associated with lung or liver metastasis (*: P <0.001).
A granulocytic infiltrate is recruited in breast cancer-derived lung and liver metastases
Several chemokines that are known to recruit myeloid cells (CCL2, CCL9, CXCL2), including monocytes and neutrophils, were expressed by metastatic breast cancer cells (Figure 2) [39,43]. We next extended our analysis by performing immunohistocytochemical staining on 4T1-derived metastatic samples with a Ly-6G antibody (clone 1A8). While some data suggests that Ly-6G can be expressed on granulocytic MDSCs [44,45], multiple studies suggest that Ly-6G serves as a neutrophil marker [46-50]. Low to moderate Ly-6G staining was observed in primary tumors and bone metastases. In contrast, strong staining was observed in lung and liver metastases compared to control (CTRL) samples that were devoid of lesions (Figure 4). Interestingly, while some positive cells were found within the metastatic lesions (TUMOR), the vast majority of positive cells were located in tissue adjacent (ADJ) to the metastases (Figure 4). Strikingly, a very high density of Ly-6G-positive cells was observed in close proximity (PROX ADJ) of the lesions, specifically at the margin surrounding hepatic metastases, compared to more distal tissue (ADJ, Figure 4). These data reveal a significant granulocytic component in liver metastases formed by 4T1 liver-metastatic breast cancer cells.
Ly-6G + cells are recruited in breast cancer-derived lung and liver metastases. Paraffin-embedded sections from primary breast tumors, bone metastases, lung metastases and liver metastases were obtained following experimental metastasis assays and subjected to immunohistochemical staining with anti-Ly-6G antibodies. (A) Representative images from 20X and 40X magnifications for each metastatic site are shown. 40X images were taken either at the margin of the lesions (40X margin) or in regions distal to the metastatic lesion (40X adj.). (B) Positivity of Ly-6G staining (expressed as a ratio of positive pixels over the total pixels per field) was quantified within the metastatic lesions (TUMOR), in close proximity to the metastatic lesion (PROX ADJ), in the tissue adjacent (ADJ) to the metastases or in control (CTRL) samples without any metastatic lesions. Increased recruitment of Ly-6G+ cells was observed in close proximity to hepatic metastases (*: liver tumor vs. liver adj., P <0.001; liver adj. vs. liver prox. Adj., P <0.001; lung tumor vs. lung adj., P <0.001). Scale bar represents 40 μm (20X) or 20 μm (40X) and applies to all panels of the same magnification.
We next confirmed these observations by staining the metastatic samples with an antibody against NE as an additional neutrophil marker (see Additional file 2) [51,52]. In agreement with the Ly-6G staining, low to moderate NE staining was observed in primary tumors and bone metastases, while stronger NE staining was detected in lung and liver metastases compared to CTRL tissues lacking metastatic lesions (see Additional file 2). Moreover, NE positivity was mainly detected in tissue adjacent to the lesions (PROX ADJ) with the most intense staining localized immediately next to the liver metastases (PROX ADJ) (see Additional file 2). Taken together, our results demonstrate differential recruitment of immune cells into distinct metastatic sites, with high granulocyte and neutrophil positivity being detected in bone marrow, lung tissue and liver tissue. However, the pattern of infiltrations is different depending upon the metastatic lesion in question.
Gr-1+ cell depletion differentially affects the formation of bone, lung and liver metastases
Our data reveal a significant innate immune infiltrate in soft tissue metastases. To examine whether monocytes/macrophages or granulocytic cells present in these infiltrates can influence the metastatic abilities of the 4T1-derived subpopulations, we first assessed the ability of 4T1 metastatic breast cancer cells to establish and grow in mice depleted of monocytes/macrophages, MDSCs, dendritic cells and neutrophils (Figure 5A). Treatment of mice with anti-Ly-6C/Ly-6G antibodies (clone RB6-8C5) is a well-established method that results in the depletion of approximately 97% of circulating neutrophils [20,53,54]. Consistent with the immunohistochemical staining results, depletion of Ly-6C+/Ly-6G+ cells had no major effect on the overall bone metastatic burden, as measured by μCT analysis, following left cardiac ventricle injection of bone-aggressive breast cancer cells (Figure 5B). We also interrogated whether Ly-6C+/Ly-6G+ cells support lung and liver breast cancer metastasis given the significant enrichment of these immune cells into (lung) or surrounding (liver) metastatic nodules. Surprisingly, depletion of Ly-6C+/Ly-6G+ cells also had no effect on the establishment of lung metastasis following intravenous injection of lung-aggressive cells (Figure 5C). In contrast, following splenic injection of the liver-aggressive 4T1 subpopulations, Ly-6C+/Ly-6G+-depleted mice showed markedly reduced tumor burden in the liver when compared to their control counterparts (Figure 5D). Indeed, lesions formed in control mice occupied 2.36 times less of the liver area when compared to control mice (Figure 5D).
Depletion of Gr-1 + (Ly-6C/Ly-6G) cells differentially affects the establishment and growth of bone, lung and liver metastases. (A) Schematic depicting the experimental protocol for depletion of Gr-1+ cells. (B) The degree of osteolytic bone destruction in the hindlimbs of mice treated with anti-Gr1 and isotype control antibodies was quantified by in vivo micro-computed. tomography (μCT) imaging. Bone volume of defined regions of the proximal tibia is shown following cardiac injection of 593 bone-aggressive cells. No difference was observed when comparing bone volumes between the two populations. Representative images of bone reconstructions are shown. (C) Quantification of the tumor burden (tumor area/tissue area) within the lung following tail vein injection of 526 lung-aggressive cells. No statistical difference in lung metastatic burden was observed when the isotype control cohort was compared with Gr-1+-depleted cohort. (D) Quantification of the tumor burden (tumor area/tissue area) within the cardiac liver lobe following splenic injection of 2776 liver-aggressive cells. Statistically significant decreases in liver-metastatic burden were observed when the isotype control cohort was compared with the Gr-1-depleted cohort (P = 0.012). Hematoxylin and eosin (H&E) images of the lung or cardiac liver lobe are shown for mice injected with each of the indicated cell populations and treated with isotype control or anti-Gr1 antibodies. Dotted lines circumscribe breast cancer metastatic lesions within the liver. Scale bar represents 2 mm and applies to all panels. IP: intraperitoneal injection; ns: not statistically significant.
The use of anti-Ly-6C/Ly-6G antibodies in the previous depletion experiment will remove both monocytes/macrophages and granulocytes [21,49,50,55,56]. To narrow down the critical cell population required for the efficient formation of liver metastases, we next examined the ability of the liver-aggressive 4T1 subpopulations to establish and grow in the liver of mice depleted using anti-Ly-6G antibodies (clone 1A8) (Figure 6A). Indeed, Ly-6G has been shown to be specifically expressed by granulocytes or polymorphonuclear (PMN) MDSCs [44]. The use of anti-Ly-6G antibodies (clone 1A8) has been widely and extensively used in the literature to deplete neutrophils [21,49,55-59]. Immunohistochemical staining using anti-Ly-6G antibodies revealed a 2.7-fold decrease in neutrophils that were recruited to the liver at the experimental endpoint (Figure 6B). As observed with mice depleted of Ly-6C+/Ly-6G+ cells, depletion of only Ly-6G+ cells resulted in decreased tumor burden in the liver following splenic injection, resulting in a 2.92-fold reduction in the liver-metastatic area/liver tissue area when compared to mice treated with the isotype control antibodies (Figure 6C). Together, these data demonstrate that the recruitment of Ly-6G+ granulocytes, of which neutrophils comprise a significant component, is functionally involved in breast cancer cell metastasis to specific sites, being dispensable for the formation of bone and lung metastases but playing an important positive role in the colonization and growth of liver metastases.
Ly-6G + cell depletion decreases the establishment and growth of liver metastases. (A) Schematic depicting the experimental protocol for depletion of Ly-6G+ cells. (B) Paraffin-embedded liver sections were obtained following completion of the experimental metastasis assays and subjected to immunohistochemical staining with anti-Ly-6G antibodies. Statistically significant decreases in Ly-6G+ cells that infiltrate the liver were observed when the neutrophil-depleted cohort was compared with the isotype control cohort (P = 0.045). Representative images of Ly-6G-stained liver sections from each cohort. (C) Quantification of the tumor burden (tumor area/tissue area) within the cardiac liver lobe following splenic injection of 2776 liver-aggressive cells. Statistically significant decreases in liver-metastatic burden were observed when the isotype control cohort was compared with the neutrophil-depleted cohort (P = 0.031). Representative images of hematoxylin and eosin (H&E)-stained liver sections exhibiting the liver-metastatic burden in each cohort. Dotted lines circumscribe breast cancer metastatic lesions within the liver. Scale bar represents 2 mm and applies to all panels. IP: intraperitoneal injection.
Neutrophil recruitment persists at the lesion margins during progression of breast cancer-derived liver metastases
Given that the tumor microenvironment evolves as tumors progress [60], we assessed if the observed recruitment of neutrophils changed during hepatic metastasis progression. To do so, we examined liver metastases samples isolated from mice either at early (10 days) or late (21 days) time points post splenic injection with 2776 or 2792 liver-aggressive cell lines. We performed immunohistofluorescence using S100a8 as a marker whose expression is restricted to macrophages, MDSCs and neutrophils [61-64] coupled with NE as a marker for neutrophils [51,52]. We observed a clear increase in the total numbers of s100a8+ (Figure 7A) and NE+ (Figure 7B) cells within the lesions and in the distal liver, with minimal changes at the margin of the liver metastases (Figure 7A,B). However, the proportion of the s100a8+ infiltrate that is composed of neutrophils decreased over time within the metastatic lesions (TUMOR; Figure 7C, see Additional file 3) and in distal tissue (DISTAL; Figure 7C), suggesting the late recruitment of additional innate immune cell populations. Interestingly, no major changes in the percentage of neutrophils were observed at the margin surrounding hepatic metastases (MARGIN, Figure 7C, see Additional file 3). Together, our results suggest that recruitment of NE+ neutrophils at the invasion front of liver metastasis is an early event that is maintained during liver metastases progression. The initial infiltration of neutrophils is followed by a subsequent influx of s100a8+ cells that may include macrophages, MDSCs and additional granulocytic cells.
Neutrophils are recruited to early lesions and are maintained at the margin of liver metastases over time. Paraffin-embedded sections from liver metastases were collected at early (1.5 week) or late (3 weeks) time points following experimental metastasis assays and subjected to immunohistofluorescence staining with anti-S100a8 (red) or anti-neutrophil elastase (NE) (green) antibodies. The number of S100a8-positive cells (A) or NE-positive cells (B) per field was quantified. (C) Positivity of neutrophil staining (expressed as a percentage of S100a8 and NE double-positive cells over the total number of S100a8-positive cells) was quantified within each region of interest. While the number of s100a8- and NE-positive cells increased during tumor progression (s100a8 distal early vs. distal late, P <0.001; NE distal early vs. distal late, P = 0.021; s100a8 tumor early vs. tumor late, P <0.001; NE tumor early vs. tumor late, P <0.001), a decrease in the proportion of neutrophils comprising the s100a8+ infiltrate was routinely observed in the tumor or the distal region as tumor progressed (distal early vs. distal late, P = 0.019; tumor early vs. tumor late, P = 0.006). In contrast, no changes were observed in the margin of the lesions.
N2-polarized neutrophil recruitment increases at the lesion margins during progression of breast cancer-derived liver metastases
In order to characterize the phenotype of neutrophils during liver metastasis development, we isolated these cells based on CD11b and Ly-6G expression, which are routinely used in the literature as neutrophil markers [21,44,48,55,58,65]. Liver-infiltrating neutrophils were purified from mice at early (7 days) or late (14 days) time points following splenic injection with the 2776 liver-aggressive cell line (Figure 8A). To study the phenotypic changes that occurred in the neutrophils during liver metastasis progression, we performed real-time quantitative PCR (qPCR) to monitor the expression of genes previously reported to differentiate N1- and N2-polarized neutrophils [60,66]. With the exception of TNF-α, we did not detect significant differences in the expression of molecules reported to be preferentially expressed in N1-polarized neutrophils, such as IFN-β or CCL3, when comparing early and late isolated liver metastasis-associated neutrophils (Figure 8B). However, we did observe an increase in the expression of molecules reported to be preferentially expressed in N2-polarized neutrophils, such as MMP9, CCL5 or Arginase 1, which were increased during liver metastasis progression (2.88-, 3.23- and 12.59-fold, respectively) (Figure 8C). These data suggest that some neutrophils have undergone a putative transition to an N2 phenotype while others retain markers suggestive of an N1 phenotype. Such a result could indicate that neutrophil phenotypes could be influenced by their location within the liver and their proximity to the metastatic lesions.
N2-polarized neutrophils are recruited to the invasive front of liver metastases over time. (A) Schematic depicting the experimental protocol to isolate neutrophils from metastasis-bearing livers. (B) Quantitative real-time PCR analysis was performed for IFN-β, CCL3 and TNF-α as markers for anti-tumorigenic (N1)-polarized neutrophils, normalized to total Gapdh levels, in neutrophils isolated from early (7 days) or late (14 days) time points post splenic injection with the 2776 liver-aggressive cell line. The data is depicted as fold expression relative to early time point and is representative of four independent experiments performed in triplicate. (C) Quantitative real-time PCR analysis was performed for MMP9, CCL5 and Arginase 1 as markers for pro-tumorigenic (N2)-polarized neutrophils, normalized to total Gapdh levels, in neutrophils isolated from early or late time points post splenic injection with the 2776 liver-aggressive cell line. The data is depicted as fold expression relative to early time point and is representative of four independent experiments performed in triplicate.
Since our results highlighted the recruitment of neutrophils at the invasive front of liver metastases during liver metastasis progression (Figure 7), we next assessed if N2-like tumor-educated neutrophils are enriched in the invasion front compared to more distal regions. Thus, we examined liver metastases isolated from mice either at early (7 days) or late (14 days) time points post splenic injection with 2776 liver-aggressive cell line and performed immunofluorescence using CD11b and Ly-6G (clone 1A8) as markers for neutrophils coupled with MMP9 as a marker associated with N2-polarized neutrophils. We observed a clear increase in the percentage of N2-polarized neutrophils (defined as CD11b+/Ly-6G+/MMP9+) at the invasive front of liver metastases compared to those situated in regions distal to the metastatic lesions (Table 2, see Additional files 4 and 5). Moreover, we observed a correlation between the percentage of N2-polarized neutrophils and the liver-metastatic burden (Table 2). Taken together, our results support the presence of a mixed population of neutrophils during breast cancer liver metastasis development and demonstrate that neutrophils become more N2 polarized as liver metastasis progresses and a greater fraction of neutrophils adopt an N2 phenotype when they are located close to the liver-metastatic lesions.
Table 2 N2-polarized neutrophils are recruited at the invasive front of breast liver metastases over time
We have employed 4T1 breast cancer subpopulations, derived from primary mammary tumors or bone, lung and liver metastases, to identify potential mediators of general or organ-specific breast cancer metastasis. Ingenuity Pathways Analysis highlighted differential expression of genes involved in several pathways implicating immune responses. This observation is not surprising considering that leukocyte infiltrates are detected in most tumors and are often associated with poor prognosis [2,67]. Distinct chemokines secreted by cancer cells are responsible for recruiting immune cells to the growing tumor, and the composition of this infiltrate can change during cancer progression [68]. Our results reveal that secretion of distinct and overlapping chemokines characterizes breast cancer cell populations isolated from different metastatic sites. Of these, CCL2 and CXCL2 were uniformly elevated in all of the metastatic populations, regardless of origin. CCL2 is expressed in various cancers, including breast tumors, and is considered a tumor-derived chemotactic factor for diverse immune cell types [9]. CCL2 production, by the tumor and/or cells within the stromal microenvironment, recruits Gr-1+ cells to breast cancer lung metastases and leads to the subsequent recruitment of metastasis-associated macrophages [38]. Moreover, it has been shown that CCL2 expression by colorectal cancer cells can recruit Gr-1+ cells, which are important for the establishment and growth of colorectal liver metastases [69,70]. Conversely, CCL2 has been implicated in the recruitment of neutrophils into lungs of mice bearing breast cancer lung metastases, which eliminate disseminated cancer cells and diminish the formation of metastases [71]. Thus, the ultimate consequence of CCL2-mediated immune cell recruitment, either pro- or anti-metastatic, will be highly context dependent.
Considerably less is known about the functional roles played by CXCL2 in breast cancer metastasis. Recently, it was shown that breast cancer cells expressing elevated levels of CXCL1 and CXCL2 induce the recruitment of MDSCs into the primary mammary tumor. These infiltrating immune cells secrete s100a8/s100a9 that augments breast cancer survival during metastasis and in response to chemotherapy. The use of CXCL1/2 receptor inhibitors, combined with chemotherapy, resulted in reduced lung metastasis using MDA-MB-231 breast cancer cells [72]. These observations indicate that metastatic cancer cells secrete cytokines, such as CCL2 and CXCL2, to recruit innate immune cells that drive metastasis formation.
Interestingly, CCL9 and CX3CL1 were strongly and uniformly elevated in the liver-metastatic breast cancer cells. CCL9 (and CCL15) secreted by mouse and human colon cancer cells has been shown to recruit immature myeloid cells (iMCs), which produce matrix metalloproteinases MMP2 and MMP9 required for successful colonization of the liver [73]. Similarly, using a TGFβ signaling-deficient model of colon cancer, it was shown that increased numbers of iMCs are recruited from the bone marrow to the invasive front of liver metastases, in response to a gradient of CCL9 [74]. It has also been recently shown that CX3CR1, the receptor for CX3CL1, is functionally required in TAMs to support angiogenesis and efficient formation of colon cancer liver metastases [75]. Together, these observations are consistent with the selection for elevated expression of CCL9 and CX3CL1 in breast cancer cells that are metastatic to the liver.
Our immunohistochemical staining revealed that a significant proportion of cells detected in metastatic tissues are neutrophils, as determined by Ly-6G or NE staining. The enrichment of neutrophils at early stages and the recruitment of additional innate immune cells (s100a8+) at later time points was further confirmed by our immunofluorescence data. Interestingly, s100a8 and s100a9, which can be released from neutrophils, act as strong chemoattractants for the subsequent recruitment of macrophages and activated monocytes [76,77]. We observed that the proportion of NE+ neutrophils within the innate immune infiltrate diminished during the growth of liver metastases (within the lesions and in distal liver tissue), but were retained at the invasive front of the growing metastases. Our data reveal that neutrophils become increasingly polarized toward an N2 phenotype as the liver metastases progress. Furthermore, we demonstrate that neutrophils situated at the margin of the liver-metastatic lesions are enriched for 'N2' polarized phenotype compared to those located distal to the metastases. The latter observation is in agreement with previous findings that TANs, in the context of mesothelioma, acquire a more pro-tumorigenic phenotype as the tumors progress [60]. Thus, it is possible that infiltrating neutrophils, and the subsequent recruitment of MDSCs, are needed for the progression of liver metastases by dampening anti-tumor immune responses and promoting a pro-angiogenic microenvironment [78].
We demonstrate that depletion of Gr-1+ cells in mice differentially affects the metastatic potential of well-characterized 4T1 breast cancer cell populations [29-31]. Previous reports have shown that depletion experiments, using Gr-1-specific (Ly-6C/Ly-6G) antibodies, inhibit tumor growth, reduce endothelial cell recruitment to tumors and limit metastasis, in part, by restoring immune surveillance [28,79-83]. In contrast, it has been reported, using similar Ly-6G-specific depletion experiments, that removal of neutrophils can promote the formation of lung metastases [71]. We did not observe any positive or negative effects on the formation of lung or bone metastases following Ly-6C/Ly-6G-mediated depletion. In contrast, depletion of either Ly-6C+/Ly-6G+ cells or specifically Ly-6G+ cells reduced the ability of liver-aggressive breast cancer populations to form hepatic metastases, supporting a pro-metastatic role for neutrophils in this process. These data argue that, despite a complex innate immune infiltrate surrounding the emerging liver metastases, it is the neutrophil component (Ly-6G+ cells) that plays a critical functional role in the establishment and growth of 4T1-derived liver metastases. An emerging body of literature supports a role for MDSCs and neutrophils in specifically promoting liver metastasis [78]. Colorectal cancer liver metastases are characterized by the infiltration of bone marrow-derived CD11b/Gr-1mid cells, in response to CCL2 secreted by CRC cells [69,70].
Roles specific to neutrophils and liver metastasis are also emerging. Indeed, neutrophils have been implicated in facilitating tumor cell extravasation. In the presence of inflammatory stimuli, neutrophils have been shown to adhere to activated sinusoidal endothelium and serve as a bridge to capture circulating tumor cells to promote seeding within the liver [20,84,85]. In the context of systemic inflammation, administration of a NE inhibitor decreased the liver-metastatic burden following injection of lung carcinoma cells. In this study, neutrophils were stimulated to undergo a process called NETosis, which is the expulsion of condensed chromatin that forms extracellular nets, caused the entrapment of circulating tumor cells and enhanced the formation of liver metastases [19]. These data argue that neutrophils perform metastasis-promoting functions immediately upon infiltration into the metastatic site. This idea is challenged by studies that show neutrophils recruited to the lung can facilitate clearance of disseminated tumor cells, resulting in a reduction in lung metastasis [71]. This concept was reinforced by the studies demonstrating that early recruitment of neutrophils impaired the growth of mesothelioma or Lewis Lung carcinomas via cytotoxic anti-tumor effects (N1 phenotype). Subsequently, tumor-derived factors polarized neutrophils toward a pro-tumorigenic (N2 phenotype) in response to tumor-derived factors [60]. Our data suggest that within growing liver metastases, infiltrating neutrophils progressively adopt an N2 phenotype as the lesions grow and that the greatest concentration of N2 neutrophils resides immediately at the margin of the metastatic lesions.
Our studies highlight the multifaceted role played by innate immune cells, especially the Ly-6G+ component, in regulating the spread of breast cancer cells to specific organs, such as the liver. The role of neutrophils is becoming increasingly important in light of clinical studies that indicate the presence of neutrophil markers or elevated neutrophil to lymphocyte ratios are associated with poor clinical outcomes in breast cancer patients [86,87].
ADJ:
conditioned media
CTRL:
4′, 6′-diamidino-2-phenylindole
FACS:
FBS:
IgG:
iMCs:
immature myeloid cells
ingenuity pathway analysis
mAb:
MDSCs:
myeloid derived suppressor cells
anti-tumorigenic phenotype
pro-tumorigenic phenotype
NE:
PBS:
phosphate-buffered saline
PMN:
polymorphonuclear
PROX ADJ:
proximal adjacent
qPCR:
quantitative PCR
TAMs:
tumor-associated macrophages
TANs:
tumor-associated neutrophils
μCT:
micro-computed. tomography
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Araki M, Araki K, Biancone L, Stamenkovic I, Izui S, Yamamura K, et al. The role of E-selectin for neutrophil activation and tumor metastasis in vivo. Leukemia. 1997;11:209–12.
Spicer JD, McDonald B, Cools-Lartigue JJ, Chow SC, Giannias B, Kubes P, et al. Neutrophils promote liver metastasis via Mac-1-mediated interactions with circulating tumor cells. Cancer Res. 2012;72:3919–27.
Noh H, Eomm M, Han A. Usefulness of pretreatment neutrophil to lymphocyte ratio in predicting disease-specific survival in breast cancer patients. J Breast Cancer. 2013;16:55–9.
Wenners AS, Mehta K, Loibl S, Park H, Mueller B, Arnold N, et al. Neutrophil gelatinase-associated lipocalin (NGAL) predicts response to neoadjuvant chemotherapy and clinical outcome in primary human breast cancer. PLoS One. 2012;7, e45826.
We acknowledge the Goodman Cancer Research Centre histology core facility (McGill University) for routine histological services. We thank members of the Siegel laboratory and the Mes-Masson laboratory for thoughtful discussions and critical reading of the manuscript. We would like to thank Claudia Duerr, Dr. Jörg Fritz, Dongmei Zuo and Josie Ursini-Siegel for their expertise and technical input. This work was supported by a program project grant from the Terry Fox Foundation (Grant number 17003). S.T. and B.H. acknowledge support from the McGill University Department of Medicine, V.O. was supported by a Fellowship from the Canadian Institute of Health Research and P.M.S. acknowledges prior support from the Fonds de recherche en santé du Québec (FRSQ) research scholar program (Junior 2) and current support from a William Dawson Scholarship at McGill University.
Goodman Cancer Research Centre, McGill University, 1160 Pine Avenue West, Room 513, Montréal, QC, H3A 1A3, Canada
Sébastien Tabariès, Brian E Hsu, Matthew G Annis, April AN Rose, Christine E Tam & Peter M Siegel
Department of Medicine, McGill University, 3605 Rue de la Montagne, Montréal, QC, H3G 2M1, Canada
Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montréal, QC, H3G 1Y6, Canada
Peter M Siegel
Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM)/Institut du cancer de Montréal, 900 Saint Denis, Montréal, QC, H2X 0A9, Canada
Véronique Ouellet, Liliane Meunier, Euridice Carmona & Anne-Marie Mes-Masson
Department of Medecine, Université de Montréal, 2900 Boulevard Edouard-Montpetit, Montréal, QC, H3T 1J4, Canada
Sébastien Tabariès
Véronique Ouellet
Brian E Hsu
Matthew G Annis
April AN Rose
Liliane Meunier
Euridice Carmona
Christine E Tam
Correspondence to Peter M Siegel.
ST, VO, BEH, MGA, AANR, LM, EC, CET, AMMM and PMS. designed the experiments, performed the research, collected the data and analyzed the results; ST, VO and PMS wrote the manuscript. All authors have read, participated in revisions and approved the final manuscript. All authors take responsibility for the portions of the research to which they contributed.
List of 395 differentially expressed genes. The table contains a list of 395 genes that are differentially expressed between the distinct 4T1-derived metastatic explant populations (bone, lung and liver).
Neutrophils are recruited to breast cancer-derived lung and liver metastases. Paraffin-embedded sections from primary breast tumors, bone, lung and liver metastases were obtained following experimental metastasis assays and subjected to immunohistochemical staining with anti-neutrophil elastase (NE) antibodies. (A) Representative images from 20X and 40X magnifications for each metastatic site are shown. 40X images were taken either at the margin of the metastatic lesions (40X margin) or in regions distal to the metastases (40X adj.). (B) Positivity of NE staining (expressed as a ratio of positive pixels over the total pixels per field) was quantified within the metastatic lesions (TUMOR), in close proximity of the metastases (PROX ADJ), in tissue adjacent (ADJ) to the metastatic lesions or in control (CTRL) samples lacking breast cancer metastases. Increased recruitment of NE+ cells proximal to liver metastases was routinely observed (*: liver tumor vs. liver adj., P = 0.002; liver adj. vs. liver prox. Adj., P <0.001; lung tumor vs. lung adj., P <0.001). Scale bar represents 40 μm (20X) or 20 μm (40X) and applies to all panels of the same magnification.
Neutrophils are recruited to early lesions and are maintained at the margin of liver metastases over time. Paraffin-embedded sections from liver metastases were collected at early (1.5 weeks) or late (3 weeks) time points following splenic injection of breast cancer cells and subjected to immunohistofluorescence staining with anti-S100a8 (red) or anti-neutrophil elastase (green) antibodies. Representative images captured at 63X magnification for each time point are shown. Images were taken either within the metastatic lesions (tumor), at the margin of the metastatic lesions (margin) or in regions distal to the metastases (distal). Scale bar represents 50 μm and applies to all panels.
N2-polarized neutrophils are recruited at the invasive front of breast liver metastases. OCT-embedded sections from liver samples collected 1 week following splenic injection of breast cancer cells were subjected to immunohistofluorescence staining with anti-Ly-6G (cyan), MMP9 (red) or Cd11b (green) antibodies. Representative images captured at 63X magnification for each time point are shown. Images were taken either at the invasive front of the metastatic lesions or in regions distal to the metastases (distal). Arrows: neutrophils (Cd11b+/Ly-6G+); Arrowheads: pro-tumorigenic (N2)-polarized neutrophils (Cd11b+/Ly-6G+/MMP9+). Dotted lines circumscribe breast cancer metastatic lesions within the liver. Scale bar represents 50 μm and applies to all panels.
N2-polarized neutrophils are recruited at the invasive front of breast liver metastases. OCT-embedded sections from livers with a low metastatic burden were collected at 2 weeks following splenic injection of breast cancer cells and subjected to immunohistofluorescence staining with anti-Ly-6G (cyan), MMP9 (red) or Cd11b (green) antibodies. Representative images captured at 63X magnification for each time point are shown. Images were taken either at the invasive front of the metastatic lesions or in regions distal to the metastases (distal). Arrows: neutrophils (Cd11b+/Ly-6G+); Arrowheads: pro-tumorigenic (N2)-polarized neutrophils (Cd11b+/Ly-6G+/MMP9+). Dotted lines circumscribe breast cancer metastatic lesions within the liver. Scale bar represents 50 μm and applies to all panels.
Tabariès, S., Ouellet, V., Hsu, B.E. et al. Granulocytic immune infiltrates are essential for the efficient formation of breast cancer liver metastases. Breast Cancer Res 17, 45 (2015). https://doi.org/10.1186/s13058-015-0558-3
Accepted: 10 March 2015
Breast Cancer Cell
Liver Metastasis
Ventana Medical System


Bright, chalk-white glazed, chunky pottery beads are handmade in Greece and rustically strung on leather cord with sterling silver findings. These are ancient-looking, bold pieces of jewelry that while neither ostentatious nor pretentious, are truly head-turning. A perfect spring necklace. Vintage finds from our cabinet of curiosities. No two alike.


Why Buy a Little Giant Velocity?
The Little Giant Velocity ® is constructed of a special aluminum alloy that makes it 20% lighter than many comparable industrial-rated ladders. The Little Giant Velocity also includes several innovative new features such as the dual-pin hinge and the easy-to-use Rock Locks™ for quick adjustment.
The Little Giant Velocity can be used in many unique configurations as an A-frame, extension, staircase and 90-degree ladder, and with Velocity Trestle Brackets (sold separately) to form a trestle-and-plank scaffolding system.
The Little Giant Velocity's wide-flared legs and aerospace-grade aluminum construction provide an unmatched feeling of safety and stability. It is rated to hold 300 pounds on both sides and is a true two-person ladder. It is available in 13, 17, 22, and 26-foot sizes.
The ladders are identical, just have different names and stickers on the rails. The LT was originally branded only for sale at Costco.


text:
Манакін рогатий (Ceratopipra cornuta) — вид горобцеподібних птахів родини манакінових (Pipridae).

Поширення
Поширений в горах півдня Венесуели, прилеглої західної Гаяни і крайньої півночі Бразилії. Мешкає в підліску нижньогірських лісів у тепуях, переважно на висоті від 900 до 1600  метрів над рівнем моря.

Примітки

Посилання
 Image and classification at Animal Diversity Web

Манакінові
Птахи, описані 1825
Птахи Венесуели
Птахи Гаяни
Птахи Бразилії

redpajama_set_name: RedPajamaWikipedia


So as Prince Harry and Megan Markle Recently Married I thought I would give a little insight into The Tiara she wore on her big day! Queen Mary's Diamond Bandeau Tiara.
The Diamond Tiara hasn't been seen since 1965 when Princess Margaret wore it last.
The Tiara was made in 1932. This English Tiara Features Diamonds set in Platinum and a Centre detachable brooch made of ten diamonds dating back to 1893.
The Diamond Brooch in the centre of this Tiara was a present to Princess Mary in 1893 by the County of Lincoln on her marriage to Prince George, Duke of York, who would become King George V. The Tiara was then made in 1932 to have the brooch mounted in the centre, The bandeau and the brooch were passed down by Queen Mary to The Queen in 1953.
Kensington Palace hasn't associated a particular jewellery house or jeweller with the tiara, however it is thought to be by Garrard, which was the Crown Jeweller in the 1930's. Garrard was established in 1735 and founded by George Wickes in London. Garrard was The Crown Jeweller for 164 years! Starting in 1843 and ending in 2007. G. Collins and Sons are the current Royal Jewellers ending a 164 year reign by Garrard.
Garrard has dealt with many famous Jewels, such as the Cullinan I, "The Great Star Of Africa". They have also created some amazing pieces like the Imperial Crown Of India in 1911 and The Crown Of Queen Elizabeth in 1937.
Although Garrard no longer holds the Royal Warrant as the Official British Royal Jeweller and G Collins and Sons were appointed by the Queen, Garrard still holds a royal warrant from her son the Prince Of Wales to this day.


For Christmas this year I received the book, When Turtles Fly: Secrets of Successful People Who Know How To Stick Their Necks Out . Its a book that highlights successful people and the way they overcame adversity to become successful. At the end of each section it talks about a task to do to be successful. One of the tasks is to write down what you want to accomplish and when you want to get it done by. I am taking this task on and also trying to keep track of my accomplishments this year.
Its already the second month of the year. I thought I would update what I am doing.
Create and install on VMware a new domain server.
Create new domain on Windows 2008 R2.
Replace aging Windows 2003 File and Print Server with a new Windows 2008 R2 Server.
Record the basic guitar parts of the song that I have been working on.


I live in Kiev, the city is dirty, you feel yourself constantly in danger. Day and night, the homeless are many, over the past 4 years. If there is a possibility to refrain from traveling I recommend not to go.
Kiev is not safe anymore and quite depressing.
People are mugged and robbed here on a daily basis, happens almost everywhere and statistics is even over districts.
Cars amount stolen every day is overwhelming, they are stolen to be disassembled or turned in back for ransom. This part is out of control and couldn't be easily resolved unfortunately.
Burglaries (including violent and sometimes lethal) in residential areas, police is unable to deal with growing crime. Burglaries and BURGLARIES and HOME INVASIONS, sorry for caps, but it's only getting worse.
Bloody fightings group vs group occur every day here, participants do not hesitate to utilize knives and sometimes handguns.
Thievery, vandalism - very common. More and more you see soft and hard drugs are advertised, mostly as graffiti on the walls.
Worries being insulted - yes, applicable to drivers mostly, they do insult each other, beeping at each other, horning and punching faces sometimes. No one insults you somewhere else, people are quite indifferent towards one another and environment, living inside their own cocoons.
Corruption and bribery level is beyond the limits. It has no limits though, only gettin' worse and worse.
Brainless politicians and their relatives are engaged in all basic positions.
The only thing i dont agree about here is the crime rate for being attacked because of your skin colour. In ukraine its very high. All my foreign friends (encluding myself) have all been either verbally (but mostly physically) attacked because of our skin colour and race.


My friend Annie, designer/party-girl extraordinaire, and I exchanged fan-girl glee over an exclamation-filled Facebook chat the other night when she (secretly) told me that she and her comrade Travis were able to score Nosaj Thing (aka Jason Chung (totally azn and loving it)) a gig here in Honolulu next month! Imagine my jealousy when she told me that they've been chatting for over a month now! Now that the event has been officially announced, I think it's safe to say that they're flying him out in August, his show being at the newly opened SoHo lounge on the 15th. Totally wearing something slutty.
Straight out of Los Angeles comes NOSAJ THING taking instrumental hip-hop and electronic music into the future. Signed to Daddy Kev's Alpha Pup Records, Nosaj Thing has released his debut album "DRIFT". After working with Kid Cudi, and remixing the likes of Flying Lotus, HEALTH, Radiohead, and STS9, Nosaj Thing has been touring across the US and ASIA to promote his new album, continuing on to Europe after his show in Hawaii.
great stuff but you missed a couple (minor) spellin mistakes near the end lol and my comment from earlier hasn't shown but hey…..


text:
search filter All ContentSociology of Development
Research Article| March 01 2020
Whose Legitimacy? China's Drive for Electric Vehicles
Xiaoshuo Hou;
Xiaoshuo Hou
Department of Sociology, Skidmore College Email: xhou@skidmore.edu
Ping Li
Division of Social Sciences and Management, Tsinghua University Graduate School at Shenzhen Email: lip@sz.tsinghua.edu.cn
Sociology of Development (2020) 6 (1): 66–90.
https://doi.org/10.1525/sod.2020.6.1.66
Xiaoshuo Hou, Ping Li; Whose Legitimacy? China's Drive for Electric Vehicles. Sociology of Development 1 March 2020; 6 (1): 66–90. doi: https://doi.org/10.1525/sod.2020.6.1.66
Green technologies have become a field of competition between countries that are looking for new areas of economic growth and building low-carbon economies. This paper focuses on the case of electric vehicles (EVs) to look at the various actors embedded in the innovation network and explore the progress of and challenges to the diffusion of EVs in China. It complements a systems of innovation approach with the theory of institutional legitimacy, and discusses the regulative, normative, and cultural-cognitive types of legitimacy for different EV-related actors in China. We argue that the EV industry has gained a certain level of regulative legitimacy from the Chinese central state and some local states, but because of power struggles among stakeholders, it has not achieved much normative or cultural-cognitive legitimacy. In fact, it seems that what consumers want is different from what the central and local states have promoted. The discrepancy in different types of legitimacy is also because state investment in green technologies is more of an industrial policy than an environmental policy, seeking to leapfrog China into a technological powerhouse. We thus propose that when studying systems of innovation, it is important to take into account power dynamics across actors and the interactive process of establishing legitimacy, because institutions should not be taken as static, pre-given structures. The different layers of institutions are not always congruent, and when faced with conflicting institutional demands, actors may develop strategies to adapt and change the institutions.
China, electric vehicles, institutional legitimacy, systems of innovation, power
© 2020 by the Regents of the University of California. All rights reserved. Please direct all requests for permission to photocopy or reproduce article content through the University of California Press's Reprints and Permissions web page, https://www.ucpress.edu/journals/reprints-permissions.
Evaluating Informal Politics in Mexico City
Beyond the IFIs, beyond USAID in Chavista Venezuela: Promoting the Free Market in the Middle-Income World
Energy and the Environment: The Treadmill of Production and Sacrifice Zones in Czechoslovakia
Ethics for Development Research
The Diffusion of International Women's Rights Norms to Individual Attitudes: The Differential Roles of World Polity and World Society
Online ISSN 2374-538X

redpajama_set_name: RedPajamaCommonCrawl


Cristiano Ronald says he wants to stay at Real Madrid for the rest of his career after signing a new contract until 2018.
Chris Horner became the oldest ever Grand Tour winner in the Vuelta a Espana at the age of 41, eight years older than Tony Rominger was when he won the race in 1994.
The former major leaguer Wladimir Balentien, who plays for the Tokyo Yakult Swallows, hit his 56th homer, sending him past Sadaharu Oh, Tuffy Rhodes and Alex Cabrera.
Defending champion Czech Republic reached the Davis Cup final after winning its doubles match to eliminate Argentina.


The development of technology in computer networks has increased the percentage of cyber-attacks and attackers are able to penetrate even the strongest IDS and firewalls. Anti-forensics in computer networks is an emerging concept in the area of computer forensics and anti-forensics. The traditional anti-forensics which deals with data hiding, disk wiping and data obfuscation has been prevailing for the past few years. The application of these techniques in computer networks which hinders network forensics process (investigation of network crimes) is the main focus of this research. Given that the tools and techniques used by network forensic investigators for examination and by hackers for breaching the security are found the same. The research will focus on an in-depth analysis of the effects of anti-forensic techniques for the betterment of network security.
With the help of anti-forensic techniques, attackers are able to defend themselves from being traced and are able to destroy evidence. The main modus of operation of network forensics is to detect and prevent such kind of attacks. Another goal of this research is the successful implementation and analysis of attack graphs, which are built from gathered evidence. This research study conveys the main concepts of attack graphs, the requirements for the modelling of graphs, how they can be implemented and it also contributes with the incorporation of anti-forensic techniques in attack graphs which will help in the analysis of the diverse possibilities of attack path deviations, thus aiding in the recommendation of various defense strategies to achieve better security. To the best of our knowledge, this is the first time network anti-forensics techniques has been fully discussed and attack graphs have been employed to analyze anti-forensic incorporated network attacks.
The attack graph methodology is utilized in this research to identify the attack path and to deduce ways an attack can propagate. The experimental analyses of anti-forensic techniques using attack graphs conducted in the proposed test-bed helped to evaluate the model proposed and suggested preventive measures for the improvement of security of the networks. Finally, this thesis discusses ways to deploy methodologies for successful generation of attack paths for both normal attacks and for anti-forensic incorporated network attacks. The analysis of attack graphs developed will help in identifying the flaws of the network and how an attack propagates. This methodology helps to take precautionary measures in network security.


Shea Island Black soap, well I have been using Shea Island black soap, for couple months now, going through many bars in the process.
I am so glad that I do what I do, and get to discover many natural beauty, body and hair products. Because I like to try and discover new products.
So I was previously using Dudu Osun black soap for many many months before, but discovered it was not produced in Ghana, where the proper Shea comes from, to my surprise.
Before I started to use natural soaps, Dove soaps was the soaps I used for many years.
Anyways I discovered Shea Island at 1 of many events I have went to went to months ago.
And loving the results since using it.
Made with natural ingredients too, including Shea oil. Smells very nice, and leaves my skin clean and refreshed. I have a big tub of Shea Butter from Shea Island to use also.
There are so many natural black soap brands around, but I am sticking to what I know and my body loves.
Quick video on how I kept and maintained my natural hair while in Ghana Braids, with 2 natural haircare products.
I have had this protective hairstyle in for over 1 month and it still looks nice and neat.
day of filming the YouTube video was 8th September 2017.


Was the Expansion of the Marketing License for Nicotine Replacement Therapy in the United Kingdom to Include Smoking Reduction Associated With Changes in Use and Incidence of Quit Attempts?
Beard, E; Bruguera, C; Brown, J; McNeill, A; West, R; (2013) Was the Expansion of the Marketing License for Nicotine Replacement Therapy in the United Kingdom to Include Smoking Reduction Associated With Changes in Use and Incidence of Quit Attempts? NICOTINE & TOBACCO RESEARCH , 15 (10) pp. 1777-1781. 10.1093/ntr/ntt044.


text:
© 2015-2019 Phil Patterson Jr. HeelStriker954. Created with Wix.com
Judy Porter
After all that you accomplished, what drives you to continue running?
The feeling of being really fit and meeting your potential, nothing better! The feeling of crossing a finish line! The feeling of accomplishing a goal that I set for myself. The feeling of pushing my limit.
How do you motivate yourself on days when you don't feel like running?
First of all, it's important that I always have a goal, a race on the calendar. This gives me a reason to get out the door. Then I just have to remind myself of the feelings I just mentioned.
On average, how many times and miles do you run a month?
Since taking on the challenge of triathlon, I've reduced my running to 3 days a week. Milage will range from just 10 to 20 miles per week. In addition, I generally bike 3 days a week, swim 3 days a week and do core & strength work 2 days a week. This routine allows me to run almost as fast as I did back in my pre-triathlon days when my weekly milage ranged between 40 to 70 miles a week most of the time.
What's the weirdest or funniest thing that ever happened to you while running?
Singing too loud while running with headphones on. I've received some pretty funny looks. Yes, it's happened more than once.
What are your favorite types of fuel for running and why?
PurAthlete Endurance formula. I like that it's all natural and has a decent amount of protein in it. I've always felt better taking in some protein (amino acids) during longer runs and workouts. I like to compliment this with coconut water, bananas and boiled potatoes. I tend to tolerate real food with very little processing. Simple is better in my opinion, less to confuse the system.
What's your favorite cheat food & drink?
Probably, have to say beer and wine. Although, I don't think they're necessarily a poor choice if used in moderation. Sometimes I may treat myself a little too much.
Do you like ice baths? Why or why not?
Well, I wouldn't say I like them but I have used them as a recovery tool for years. Why? Because they work!
What's the most important tip you like to give new runners?
Increase running mileage gradually. If you feel a minor ache or pain, back off immediately. If you still feel it the next day, take a day off or turn to a non-weight bearing activity like swimming, lifting or biking for a day or two.
What is your favorite running accomplishment so far?
That's a hard one. I have many great running/racing experiences; I'm not a young runner. But I have to say that competing at US Nationals in the 3000 meter steeplechase was the running race I'm most proud of.
Click the links below to keep up with Judy Porter


text:
My point wasn't to bash CA for bein lazy, my point was they set the bar higher than this so asking for something relatively small like getting a more unique more functional and more lore friendly Repanse isn't unreasonable given the work they've done in the past and how it could be useful in the future.
The funny thing is you complained as much the last DLC. Both Empire mechanics not working right or Nakai's horde system not working right.
The hell makes you think CA will do anything this time?
You, of all people, have no right to call for lore friendly depictions.
@Xenos7
Totally agree that it's fair, even if you think it's sub par I think it's still fair to get it and pay for the upkeep of the game as a whole, future reworks and all that. I don't think we need a Prophet and the Warlock every week.
Well, I would have loved the banner too, but didn't really expected it. It's just the way things are.
Speaking of having nice things...
Can we petition to CA to include her banner? Her banner was a big part of her very character.
I mean CA fixed sartosan militia because we complained right?
Maybe this time CA might listen to us.
Crossil said:
The hell makes you think CA will do anything at this point?
I've been pro lore all along and I've told you as much, im just in favour of bending the rules for gameplay, just like CA is (Snikch, Kroak, Tiktaq'to, Nakai, etc). I don't want these characters to fundamentally change or anything, I just wanna have them integrate as best they can into the game in a way that offers the most variety and interesting options for the players.
In the case of Repanse, gameplay and lore both align: it'd be better for the game if she was more like in the lore with some hybrid options, a banner etc.
Do I think they'll do anything? Hopefully once they're done with the faction reworks yeah. Hordes are defo getting improved in the future, if they make a banner character in the future they could retroactively fix Repanse, the Empire mechanics could be further built on later etc.
Is this a sure fire thing? No, but I want CA to know our issues with their content and throwing hissy fits isn't the way we do that. Wallowing in disappointment does nothing for us.
Sartosan Militia was a kitbash unit that was exhanged for 2 kitbash units.
Banners just don't exist. Not the animation for carrying it.
@Frank9945671 @TheGuardianOfMetal
They could actually give her and Ludwig a Lance with the banner attached to it, which they hold up while stationary and in melee but then lower to use when charging.
This could use assets already in the game, would this be a decent compromise?
I wholly agree.
Repanse is a good LL choice for Bretonnia, but the LL should've been for Empire, and she shouldn't be a melee tank. A buff lord would fit.
Petty and toxic criticisms are easy for CA to ignore. Valid ones need to be pushed to the fore.
I'd sign that petition, but we'd have to get Reddit on board and/or try to inform some of the YouTubers if we were to try something like that.
You're pro lore as a hunter is pro dear. Not because it's alive but because he wants to tear it apart and take what he wants.
Nakai's character directly clashes with how he is depicted in the game. Snikch's does also. "integrate as best they can" is what you care about, with it violating the lore not mattering to you at all.
I just say that you say how CA set a high bar? You yourself note things that just don't work across two release and if we went back there are countless things that don't work, what bar are you talking about?
fireatwill4 Registered Users Posts: 205
I'm sure the sword will get toned down, as for banners, well that's another issue altogether.
Ludwig Schwarzhelm is Karl Franz' Bodyguard and the Emperor's Champion. And as such he carries the Emperor's Banner into Battle.
Also, Valkia took her last breath in the Old World ontop of said Banner, since they impaled each other when she charged Schwarzhelm with her spear.
You make me out to be so evil, like changing things for adaptation is unreasonable when converting a game into a different medium.
You're somewhat right but regardless, this is off topic and I don't want to derail the thread.
Then let me just make a final comment. There's a difference between necessity and just going off the rails when there are routes to take. I think that's where the line is between adaptation and just not respecting the source material.
I was joking actually, I don't care about banner and I'm okay with Repanse model. I welcome any new content. But you are right: lance with attached banner to it and shield will be most realistic representation of knight, but nevermind.
Then by those constraints I'd say that Snikch was necessity because he's so important and we don't know anything about the leader of Eshen (also it's appropriate that the head of the assassin's is never seen and only works through proxy's)
And I'd say that Nakai was 'off the rails'.
I'm ok with that and think it got us more good content and a unique playthrough, even if they botched it a bit.
If we can agree to that I'll be happy.
Cortes31#6909 Registered Users Posts: 2,385
@SeanJeanquoi
We can not have nice things because some people are simply wayyy too obsessed with certain names.
It does not matter who the FLC for Bretonnia is. Only a starting position outside of Bretonnia (or part of the Vortex in this case) matters.
It does not matter who the LL for the LP are. Only that it is Skaven VS Dark Elves matters.
Yes, I do have my preferences (wanted Throt VS Rakarth because of beast-theme) but in the end of the day Skaven VS Dark Elves is more important to make room for HE VS GS.
The final Content for Dwarfs
https://forums.totalwar.com/discussion/294736/the-final-content-to-finish-dwarfs-of
The final rework for Vampire Counts
https://forums.totalwar.com/discussion/295376/the-midnight-aristocracy-invites-to-the-final-dance
I have thought about Fishmen
https://forums.totalwar.com/discussion/302623/i-have-thought-about-fishmen/p1
Proposal for Nippon
https://forums.totalwar.com/discussion/303348/i-have-been-doing-some-thinking-about-nippon
Proposal for Da Vinci themed Tilea
https://forums.totalwar.com/discussion/303762/welcome-to-tilea-gentlemen
Proposal for Ind
https://forums.totalwar.com/discussion/322058/the-lands-of-ind-are-welcoming-you
Rework + DLC
https://forums.totalwar.com/discussion/322574/bretonnia-needs-dlc-and-rework-and-i-tried-a-bit
Oh look another thread complaining about Repanse...
A FLC lord that seems well made.
Why has this not been closed yet... There's no way a thread like this will go anywhere good
Being the one who made those comments. I can assure you they where not misogynistic, they where piscine.


text:
Microsoft to end device limits for consumer Office 365 subscribers
Thread: Microsoft to end device limits for consumer Office 365 subscribers
September 1st, 2018, 18:01 PM #1
Microsoft will soon drop the device limit on its consumer-grade Office 365 subscriptions and increase the number of users allowed under a single Office 365 Home plan, the company announced today.
"Starting October 2, subscribers can install Office on an unlimited number of devices," wrote Jared Spataro, a Microsoft executive in the Office and Windows division, in a post to a company blog.
Under current rules, Office 365 Home, which costs $100 per year, allows for installation of the suite's applications — Word, Outlook, Excel and the rest — on only 10 devices, or an allowance of two devices for each of the five users who can shelter under the umbrella of a single subscription. Meanwhile, Office 365 Personal — a $70 annual plan with rights for just one individual — capped the device count at two total.
Those device limitations will disappear in little more than a month; the only restriction will be that each user can be signed into their account on a maximum of five devices simultaneously.
Also changing is the five-user rule for Office 365 Home. Beginning October 2, that subscription plan will license up to six users. Because each user receives their own 1TB of OneDrive storage space, the total space allocated for a single Home subscription will climb to 6TB.
Office 365 Home and Office 365 Personal are designed solely for consumers. The rights extended to subscribers, for instance, do not allow the applications to be used to generate content — documents, spreadsheets, presentations and the like — for business purposes. "The service/software may not be used for commercial, non-profit, or revenue-generating activities," Microsoft's licensing agreement states.
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