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Formyl peptide receptors (FPR1, FPR2 and FPR3) are innate immune sensors of pathogen and commensal bacteria and have a role in colonic mucosa homeostasis. We identified FPR1 as a tumour suppressor in gastric cancer cells due to its ability to sustain an inflammation resolution response with antiangiogenic potential. Here, we investigate whether FPR1 exerts similar functions in colorectal carcinoma (CRC) cells. Since it has been shown that the commensal bacterium Lactobacillus rhamnosus GG (LGG) can promote intestinal epithelial homeostasis through FPR1, we explored the possibility that it could induce proresolving and antiangiogenic effects in CRC cells. We demonstrated that pharmacologic inhibition or genetic deletion of FPR1 in CRC cells caused a reduction of proresolving mediators and a consequent upregulation of angiogenic factors. The activation of FPR1 mediates opposite effects. Proresolving, antiangiogenic and homeostatic functions were also observed upon treatment of CRC cells with supernatant of LGG culture, but not of other lactic acid or nonprobiotic bacteria (i.e. Bifidobacterium bifidum or Escherichia coli). These activities of LGG are dependent on FPR1 expression and on the subsequent MAPK signalling activation. Thus, the innate immune receptor FPR1 could be a regulator of the balance between microbiota, inflammation and cancer in CRC models.

Additional file 6: Supplementary Figure 5. Immunohistochemical evaluation of 8505c xenografts. A. Proliferation index (Ki-67) assessed by immunohistochemistry of 8505c pCMV3 and pCMV3 PD-1 cl13 xenografts harvested 28 days post-inoculation. Representative images are shown. B. Proliferation index (Ki-67) assessed by immunohistochemistry of 8505c xenografts harvested 35 days post-inoculation in mice treated with Nivolumab or control IgG4. Representative images are shown.

Additional file 3: Supplementary Figure 2. Functional activity of intrinsic PD-1 circuit in TC cells. A. Expression levels of PD-1 in 8505c and TPC1 cells or in 8505c and TPC-1 transiently transfected with pFLAG or pFLAG PD-1, assessed by western blot. A representative experiment is shown. B. Cell cycle distribution of 8505c and TPC-1 cells transiently transfected with pFLAG or pFLAG PD-1, measured by Propidium Iodide (PI) staining by means of Flow Cytometry. The percent of the cells distributed in G0/G1, S, G2/M was indicated in each panel. Representative experiments are shown. C. Percent of apoptotic cells assessed by TUNEL reaction in 8505c and TPC-1 cells transiently transfected with pFLAG or pFLAG PD-1 and treated or not with soluble PD-L1 (sPD-L1 - 1 μg/ml). Data are presented as mean ± SD of 5 independent experiments. D. Cytofluorimetric evaluation of PD-1 expression in 8505c cells treated with siPD-1 (solid lines) or scrambled siCTR (dotted line) (100 nM). A representative experiment is shown. E. Cell cycle distribution of 8505c and TPC-1 cells treated with Nivolumab (Nivo - 10 μg/ml) or control IgG4 (10 μg/ml), measured by Propidium Iodide (PI) staining by means of Flow Cytometry. The percent of the cells distributed in G0/G1, S, G2/M was indicated in each panel. Representative experiments are shown. F. Percent of apoptotic cells assessed by TUNEL reaction in 8505c and TPC-1 cells treated with siPD-1 (100 nM) or Nivolumab (Nivo - 10 μg/ml) or the relative controls. Data are presented as mean ± SD of 5 independent experiments. G. DNA synthesis of 8505c cells transiently transfected with pCMV3, pCMV3 PD-L1 or pCMV3 PD-L2 or treated with anti-PD-L1, anti-PD-L2 blocking antibodies or IgG1 isotype control (10 μg/ml) assessed by BrdU incorporation. Data are presented as mean ± SD of 5 independent experiments. * P

Additional file 2: Supplementary Figure 1. Immune checkpoint expression in thyroid cancer (TC) cells. Protein expression levels assessed by western blot (A) and mRNA expression indicated as ΔCt for PD-1, PD-L1 and PD-L2 (B) in H-6040 normal thyroid epithelial cells, PTC-derived cell lines (BcPAP and TPC-1), and ATC-derived cell lines (8505c, CAL62, SW1736, FRO, BHT101, HTH7, OCUT1). A representative western blot experiment is shown. PCR data are presented as mean ± SD of 5 independent experiments.

Additional file 5: Supplementary Figure 4. Effects of intrinsic PD-1 on SHP2 localization and functions. A. Expression levels of PD-1, phospho-PD-1, SHP2 and phospho-SHP2 in 8505c and TPC-1 cells transiently transfected with pFLAG PD-1 or the empty vector pFLAG, assessed by western blot. A representative experiment is shown. B. Immunofluorescence microscopy of 8505c cells, transiently transfected with pFLAG PD-1 or the empty vector, with antibody specific for SHP2. Arrows indicate the surface signal of SHP2. Bars, 5 μm. A representative experiment is shown. C. Immunofluorescence microscopy of 8505c cells transiently transfected with pCMV6 PD-1-GFP and stained with antibody specific for SHP2, and the merged signal. Arrows indicate the surface signal of SHP2. Bars, 5 μm. A representative experiment is shown. D. Total protein extracts from TPC-1 cells transiently transfected with pFLAG-PD-1 or the empty vector pFLAG were subjected to a pull-down assay using the indicated recombinant proteins or to immunoprecipitation using the indicated antibodies. Proteins were immunoblotted with antibody against SHP2 or GRB2. A representative experiment is shown. E. Total cell protein extracts from 8505c cells transiently transfected with combination of pCEFL H-Ras AU5, pFLAG PD-1 or empty vector (pFLAG + pCEFL) were subjected to immunoprecipitation with anti-phospho tyrosine followed by western blotting with pan (RAS) antibody. A representative experiment is shown, together with the mean densitometric analysis ± SD of 5 independent assays. * P

Additional file 4: Supplementary Figure 3. Signalling pathways downstream PD-1overexpression. A. Expression levels of PD-1 in some clones or mass populations obtained from 8505c cells stably transfection with PD-1, assessed by western blot. A representative experiment is shown. B. Expression levels of phosphorylated forms of BRAF, MEK1/2 and MAPK (p44/p42) in 8505c cells stably transfected with PD-1 or the empty vector, assessed by western blot. A representative experiment is shown. C. Activation of AKT, SRC, S6, S6K, 4EBP1 in 8505c and TPC-1 cells, transiently transfected or not with PD-1 or the relative empty vector, assessed by western blot for their phosphorylated forms. A representative experiment is shown.

Additional file 1: Supplementary Table 1. Mouse immune cell density (expressed as percentage of CD45+ leukocytes) in 8505c xenografts.

Non-resolving inflammation is an enabling feature of cancer. A novel super-family of lipid mediators termed Specialized Pro-resolving Mediators (SPMs) have a role as bioactive molecules mediating the resolution of inflammation in cancer biology. SPMs are derived from ω-3 and ω-6 polyunsaturated fatty acids through the activity of lipoxygenases. SPMs have been described to directly modulate cancer progression by interfering with the epithelial to mesenchymal transition and invasion of cancer cells. SPMs have also been demonstrated to act on several components of the tumor microenvironment (TME). Consistently with their natural immunomodulatory and anti-inflammatory properties, SPMs are able to reprogram macrophages to favor phagocytosis of cell debris, which are an important source of pro-inflammatory and pro-angiogenic signals; sustain a direct cytotoxic immune response against cancer cells; stimulate neutrophils anti-tumor activities; and inhibit the development of regulatory T and B cells, thus indirectly leading to enhanced anti-tumor immunity. Furthermore, the resolution pathways exert crucial anti-angiogenic functions in lung, liver, and gastrointestinal cancers, and inhibit cancer-associated fibroblast differentiation and functions in hepatocellular carcinoma and pancreatic cancer. The present review will be focused on the potential protective effects of resolution pathways against cancer, exerted by modulating different components of the TME.

Background The programmed cell death-1 (PD-1) receptor and its ligands PD-L1 and PD-L2 are immune checkpoints that suppress anti-cancer immunity. Typically, cancer cells express the PD-Ls that bind PD-1 on immune cells, inhibiting their activity. Recently, PD-1 expression has also been found in cancer cells. Here, we analysed expression and functions of PD-1 in thyroid cancer (TC). Methods PD-1 expression was evaluated by immunohistochemistry on human TC samples and by RT-PCR, western blot and FACS on TC cell lines. Proliferation and migration of TC cells in culture were assessed by BrdU incorporation and Boyden chamber assays. Biochemical studies were performed by western blot, immunoprecipitation, pull-down and phosphatase assays. TC cell tumorigenicity was assessed by xenotransplants in nude mice. Results Human TC specimens (47%), but not normal thyroids, displayed PD-1 expression in epithelial cells, which significantly correlated with tumour stage and lymph-node metastasis. PD-1 was also constitutively expressed on TC cell lines. PD-1 overexpression/stimulation promoted TC cell proliferation and migration. Accordingly, PD-1 genetic/pharmacologic inhibition caused the opposite effects. Mechanistically, PD-1 recruited the SHP2 phosphatase to the plasma membrane and potentiated its phosphatase activity. SHP2 enhanced Ras activation by dephosphorylating its inhibitory tyrosine 32, thus triggering the MAPK cascade. SHP2, BRAF and MEK were necessary for PD-1-mediated biologic functions. PD-1 inhibition decreased, while PD-1 enforced expression facilitated, TC cell xenograft growth in mice by affecting tumour cell proliferation. Conclusions PD-1 circuit blockade in TC, besides restoring anti-cancer immunity, could also directly impair TC cell growth by inhibiting the SHP2/Ras/MAPK signalling pathway.

Pluripotency confers Embryonic Stem Cells (ESCs) the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives, producing the majority of cell types. Although the majority of ESCs divide without losing pluripotency, it has become evident that ESCs culture consists of multiple cell populations with different degrees of potency that are spontaneously induced in regular ESC culture conditions. Zscan4, a key pluripotency factor, marks ESC subpopulation that is referred to as high-level of pluripotency metastate. Here, we report that in ESC cultures treated with retinoic acid (RA), Zscan4 ESCs metastate is strongly enhanced. In particular, we found that induction of Zscan4 metastate is mediated via RA receptors (RAR-alpha, RAR-beta, and RAR-gamma), and it is dependent on phosphoinositide-3-kinase (PI3K) signaling. Remarkably, Zscan4 metastate induced by RA lacks canonical pluripotency genes Oct3/4 and Nanog but retained both self-renewal and pluripotency capabilities. Finally we demonstrated that the conditional ablation of Zscan4 subpopulation is dispensable for both endoderm and mesoderm but is required for ectoderm lineage. In conclusion, our research provides new insights about the role of RA signaling during ESCs high pluripotency metastate fluctuation.