Efferocytosis produces a prometastatic landscape during postpartum mammary gland involution

Abstract

qRT-PCR. Whole-tumor RNA was harvested with an RNeasy kit (QIAGEN), and cDNA was synthesized (High Capacity; Applied Biosystems) and amplified using the murine cDNA-specific primers (Integrated DNA Technologies) listed in Supplemental Methods, along with SYBR Green Supermix (Bio-Rad). The following primers were used: MRC1 (forward: 5′ CCCTCAGCAAGCGATGTGC 3′; reverse: 5′-GGATACTTGCCAGGT CCCCA-3′); iNOS (forward: 5′-GGAGCATCCCAAGTACGAGTGG-3′; reverse: 5′-CGGCC-CACTTCCTCCAG); IL10 (forward: 5′-GGCGCTGTCATC-GATTTCTCC; reverse: 5′-GGCCTTGTAGACACCTTGGTC); Tgfb1 (forward: 5′-CGCAACAACGCCATCTATGAG; reverse: 5′-CGG-GACAGCAATGGGGGTTC); IL4 (forward: 5′-GGTCACAGGAGAAGG-GACG; reverse: 5′-GCGAAGCACCTTGGAAGCC);, IL12b (forward: 5′-GGAGTGGGATGTGTCCTCAG; reverse: 5′-CGGGAGTCCAGTC-CACCTCT); CCL3 (forward: 5′-CCACTGCCCTTGCTGTTCTTCTCT; reverse: 5′-GGGTGTCAGCTCCATATGGCG); and Rplp0 (forward: 5′-TCCTATAAAAGGCACACGCGGGC; reverse: 5′-AGACGATGT-CACTCCAACGAGGACG). Target To generate apoptotic MCF7, cells were treated in suspension with 1 μm BKM120 plus 2 μm ABT-263 (both inhibitors from Selleck Chemicals) for 4 hours, washed 5 times with PBS to remove residual drug, and used directly for efferocytosis assays or for annexin V staining. For efferocytosis coculture assays, Raw264.7-GFP cells (10 4 /well) and PyVmT or MCF7 cells (72 hours after infection with Ad.mCherry and Ad.HS-V-TK) were seeded together in a monolayer in 24-well plates in 2% FBS and cultured for 24 hours prior to the addition PBS or gancyclovir. Cells were imaged at 8, 16, and 32 h after addition of gancyclovir. Cells were collected and counted under fluorescence after 32 hours of coculture. In some experiments, Raw264.7-GFP cells (10 4 / well) were seeded in a monolayer in 24-well plates and cultured for 24 hours prior to the addition of 10 3 live MCF7-mCherry or 10 3 dead MCF7-mCherry cells in serum-free media. Where indicated in the figures, BMS-777607 (1 μm) or a neutralizing goat anti-mouse MerTK antibody (AF591, 25 μg/ml; R&D Systems)(44) was added 2 hours prior to the addition of gancyclovir or 2 hours prior to the addition of dead MCF7 cells to macrophage monolayers. Live and dead MCF7 cells were similarly seeded without Raw264.7 cells as single cultures. Media were collected after 16 hours of coculture, passed through a 0.2-μm filter, and used neat (250 μl) to quantify murine IL-10 and IL-4 by ELISA (BioLegend) according to the manufacturer's protocol. Total remaining cells were collected after 16 hours of coculture, lysed, and RNA was collected using an RNeasy kit (QIAGEN). MethodsMice. All mice were inbred on an FVB background for more than 10 generations. WT FVB, MMTV PyVmT and MerTK -/-mice (67), originally referred to as Mer KD , were purchased from The Jackson Laboratory. Mice were genotyped by PCR of genomic DNA as previously described(30). Female virgin mice were randomized into 2 groups: (a) 1 group that remained virgin, and (b) 1 group that was bred from 42 to 44 days of age with WT male mice. Pregnancies were timed according to identification of a va...

Apoptosis-induced CXCL5 accelerates inflammation and growth of prostate tumor metastases in bone

“…Furthermore, activation of Stat3 by efferocytosis mediates the M2-like polarization of bone marrow macrophages (12). In vivo studies in breast and colon cancer showed that efferocytosis induced M2 polarization and metastasis, while increased cell death correlated with poorer outcomes (13,48). Moreover, the proinflammatory cytokine IL-6 induced M2-like macrophage polarization in human macrophages (49).…”

“…Phagocytosis of apoptotic cancer cells (termed efferocytosis) is a process often underestimated during tumor progression due to rapid clearance by phagocytes (11). Tumor cell death, significantly amplified by chemotherapies or other targeted therapies, triggers extensive efferocytosis, which has been suggested to accelerate further tumor growth at least in part by inducing M2-like macrophage polarization and resultant protumoral functions (12,13).…”

A fate worse than death: apoptosis as an oncogenic process

“…Besides promoting tumour growth, apoptotic cells may also facilitate metastatic tumour progression 69 . It is well known that pregnancy reduces the overall risk of developing breast cancer; however when breast cancer is diagnosed in postpartum women it is often more aggressive and has a poorer prognosis compared to disease diagnosed in nulliparous women 70 .…”

“…Implicating the clearance of apoptotic cells in cancer progression, a recent study in an MMTV-PyMT mouse model of breast cancer found that a high level of efferocytosis in parous animals promoted TAM infiltration, stimulation of a wound-healing cytokine response and increased metastasis 69 .…”

Alive failures behind the windows of cancer therapy

“…Besides promoting tumour growth, apoptotic cells may also facilitate metastatic tumour progression. Furthermore, through massive cell death, leading to extensive efferocytosis (clearance of dead cells), promoted TAM infiltration, stimulation of a wound-healing cytokine response and increase in metastasis, implicate the clearance of apoptotic cells in cancer progression (Stanford et al 2014). Controlling of caspase activity is another important fact, as the level of caspase activity required to kill a cell is not so high (Rehm et al 2006).…”