Hepatocytes paradoxically affect intrahepatic IFN-γ production in autoimmune hepatitis due to Gal-9 expression and TLR2/4 ligand release.

Hepatocytes are the targets in autoimmune hepatitis (AIH) that results in T cell-dependent liver injury. However, hepatocytes may also affect the hepatic T cells in AIH, but the underlying mechanisms are not fully understood. Here we report that hepatocytes could secrete galectin-9 (Gal-9) to suppress the intrahepatic production of Th1 cytokine IFN-γ and restrict AIH development, but hepatocyte damage resulted in opposite effects due to release of TLR2/4 ligands that promoted the intrahepatic production of IL-1ß, IL-6, and IL-12. Through Tim-3, Gal-9 could efficiently suppress the intrahepatic T cell activation despite presence of TLR2/4 ligands, thus attenuating Th1 response in AIH. Intriguingly, intrahepatic IL-6/IL-12 suppressed the effect of TGF-ß on Treg cells. Therefore, in AIH, Gal-9 promoted Foxp3 expression and function of hepatic Treg cells through TL1A signaling, although Treg function was still impaired, compared with that in naive state. Due to its promoting effect on Treg function, together with its effect on T effector cells in a Tim-3-independent way, Gal-9 could attenuate intrahepatic IFN-γ production by hindering the increase of hepatic CD4+CD43+ T cells resulting from extrahepatic T cell activation. TLR2/4 ligands attenuated the effects of Gal-9 on Treg cells and CD4+CD43+ T cells by increasing intrahepatic IL-6 and IL-12. Blocking TLR2/4 ligands could efficiently suppress intrahepatic IFN-γ production, liver injury, and hepatic fibrosis. These findings suggest that hepatocytes paradoxically affect Th1 response in AIH due to Gal-9 expression and TLR2/4 ligands release, and that targeting TLR2/4 signaling may provide an important approach in the therapeutic strategy for AIH.

Chemical induced inflammation of the liver breaks tolerance and results in autoimmune hepatitis in Balb/c mice.

Autoimmune hepatitis (AIH) is a chronic liver disease mediated by immunity, and could lead to liver fibrosis and hepatocellular carcinoma. However, the mechanisms for breaking hepatic tolerance and driving AIH still remain elusive. We herein reported that the non-specific liver inflammation triggered by carbon tetrachloride (CCl4) recruited high numbers of CD4+T, CD8+T and B cells, and elevated the expression of proinflammaitory cytokines in Balb/c mice, further breaking liver tolerance and inducing autoimmune response, AIH inflammation and liver fibrosis in the presence of CYP2D6 antigen mimicry. In contrast, adenovirus infection could not break liver tolerance and induce AIH in Balb/c mice even in the presence of CYP2D6 antigen mimicry. These results suggested that genetic predisposition could determine liver tolerance in Balb/c mice. The chemical induced inflammation in the liver breaks tolerance and might be considered important for the initiation and development of AIH in Balb/c mice.

TGF-ß1 and TNF-α synergistically induce epithelial to mesenchymal transition of breast cancer cells by enhancing TAK1 activation.

TGF-ß1 is a main inducer of epithelial to mesenchymal transition (EMT). However, many breast cancer cells are not sensitive to the EMT induction by TGF-ß1 alone. So far, the mechanisms underlying the induction of TGF-ß1-insensitive breast cancer cells remains unclear. Here we report that TNF-α can induce EMT and invasiveness of breast cancer cells which are insensitive to TGF-ß1. Intriguingly, TGF-ß1 could cooperate with TNF-α to promote the EMT and invasiveness of breast cancer cells. The prolonged co-stimulation with TGF-ß1 and TNF-α could enhance the sustained activation of Smad2/3, p38 MAPK, ERK, JNK and NF-κB pathways by enhancing the activation of TAK1, which was mediated by the gradually up-regulated TßRs. Except for JNK, all of these pathways were required for the effects of TGF-ß1 and TNF-α. Importantly, the activation of p38 MAPK and ERK pathways resulted in a positive feed-back effect on TAK1 activation by up-regulating the expression of TßRs, favoring the activation of multiple signaling pathways. Moreover, SLUG was up-regulated and required for the TGF-ß1/TNF-α-induced EMT and invasiveness. In addition, SLUG could also enhance the activation of signaling pathways by promoting TßRII expression. These findings suggest that the up-regulation of TßRs contributes to the sustained activation of TAK1 induced by TGF-ß1/TNF-α and the following activation of multiple signaling pathways, resulting in EMT and invasiveness of breast cancer cells.

IL-37 suppresses the sustained hepatic IFN-γ/TNF-α production and T cell-dependent liver injury.

T cell-dependent liver injury is an important reason for the massive hepatic damage and cirrhosis. So far it is unclear whether the development of the disease could be efficiently suppressed by anti-inflammatory cytokine that modulates innate immune cells. Here we report that anti-inflammatory cytokine IL-37 could efficiently suppress the sustained hepatic expression of IFN-γ and TNF-α, two critical cytokines for inducing hepatocyte apoptosis and liver fibrosis in T cell-dependent liver injury. IL-37 could directly suppress IFN-γ/TLR4 ligand-induced M1 activation of macrophages, thus reducing the expression of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-12. Moreover, IL-37 attenuated Th1 response in vivo and increased the expression of Th2 cytokines IL-4 and IL-13, which in turn promoted M2 activation of macrophages in the liver. The increase of M2 activation not only further reduced TNF-α, IL-1ß and IL-12 expression, but also increased IL-10 and IL-1Ra expression in macrophages, thus more efficiently suppressing the hepatic IFN-γ expression. By suppressing IFN-γ/TNF-α expression, IL-37 suppressed the up-regulation and activation of MLKL that drives hepatocellular necrosis in T cell-dependent liver damage. Accordingly, IL-37 efficiently reduced liver injury and hepatic inflammation after the repeated ConA challenge and the induction of autoimmune hepatitis, and also suppressed hepatic fibrosis resulting from the sustained liver damage. This study showed that the direct and indirect effect of IL-37 on macrophages could reduce the hepatic TNF-α expression, and also modulate IL-1ß/IL-12 and IL-10/IL-1Ra expression to suppress the hepatic IFN-γ expression, thus suppressing the development of T cell-dependent liver injury such as autoimmune hepatitis.

TLR2/4 ligand-amplified liver inflammation promotes initiation of autoimmune hepatitis due to sustained IL-6/IL-12/IL-4/IL-25 expression.

Autoimmune hepatitis (AIH), a serious autoimmune liver disease, can be a lifelong illness, leading to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). So far the mechanisms for disease initiation are largely unknown. Here we report that the amplified non-AIH liver inflammation could promote the initiation of AIH due to the sustained increase of IL-6, IL-12, IL-4, and IL-25 in the liver. The liver injury resulting from virus (adenovirus) or chemicals (CCl4) could induce an amplified (stronger/long-lasting) hepatic inflammation by releasing the ligands for TLR2/TLR4. The amplified inflammation resulted in the increase of multiple cytokines and chemokines in the liver. Among them, the sustained increase of IL-6/IL-12 resulted in the activation of STAT3 and STAT4 in hepatic CD4+CD25+ Treg cells, thus suppressing Foxp3 gene expression to reduce the suppressive function of Treg cells in the liver, but not those in the spleen. The increase of IL-12 and the impairment of Treg function promoted Th1 response in presence of self-mimicking antigen (human CYP2D6). Intriguingly, the amplified inflammation resulted in the increase of IL-4 and IL-25 in the liver. The moderate increase of IL-4 was sufficient for cooperating with IL-25 to initiate Th2 response, but inefficient in suppressing Th1 response, favoring the initiation of autoimmune response. Consequently, either adenovirus/CYP2D6 or CCl4/CYP2D6 could induce the autoimmune response and AIH in the mice, leading to hepatic fibrosis. The findings in this study suggest that the amplified non-AIH inflammation in the liver could be a driving force for the initiation of autoimmune response and AIH.

C/EBPß enhances platinum resistance of ovarian cancer cells by reprogramming H3K79 methylation.

Chemoresistance is a major unmet clinical obstacle in ovarian cancer treatment. Epigenetics plays a pivotal role in regulating the malignant phenotype, and has the potential in developing therapeutically valuable targets that improve the dismal outcome of this disease. Here we show that a series of transcription factors, including C/EBPß, GCM1, and GATA1, could act as potential modulators of histone methylation in tumor cells. Of note, C/EBPß, an independent prognostic factor for patients with ovarian cancer, mediates an important mechanism through which epigenetic enzyme modifies groups of functionally related genes in a context-dependent manner. By recruiting the methyltransferase DOT1L, C/EBPß can maintain an open chromatin state by H3K79 methylation of multiple drug-resistance genes, thereby augmenting the chemoresistance of tumor cells. Therefore, we propose a new path against cancer epigenetics in which identifying and targeting the key regulators of epigenetics such as C/EBPß may provide more precise therapeutic options in ovarian cancer.

IL-35 induces N2 phenotype of neutrophils to promote tumor growth.

IL-35 is an immunosuppressive cytokine and exerts regulatory effects on T cells, B cells, macrophages and dendritic cells. Neutrophils are important innate immune cells that play key roles in tumor development. The effect of IL-35 on neutrophils remains unknown. Here, we report that IL-35 can induce N2 neutrophil polarization (protumor phenotype) by increasing G-CSF and IL-6 production, and promote neutrophil infiltration into tumor microenvironment. The sustained expression of IL-35 could promote chronic inflammation to augment the proangiogenic and immunosuppressive function of neutrophils. IL-35 stimulated macrophages to secrete proinflammatory cytokines IL-1ß and IL-6. IL-1ß stimulated γδ T cells to produce IL-17, which in turn increased the production of G-CSF. By increasing the expression of G-CSF and IL-6, IL-35 could up-regulate the expression of MMP-9 and Bv8, and down-regulate TRAIL expression in neutrophils, thus augmenting the proangiogenic function of neutrophils. Moreover, G-CSF/IL-6 induced the enhanced activation of STAT3 and ERK pathways in neutrophils, thus increasing the expression of iNOS to suppress T cell activation. Our findings suggest that IL-35 can promote tumor progression by functioning as an up-stream cytokine to promote cancer-associated inflammation and control neutrophil polarization. Targeting IL-35 might be an important approach for designing new strategy of tumor therapy.

Circulating tumor cells promote the metastatic colonization of disseminated carcinoma cells by inducing systemic inflammation.

Circulating tumor cells (CTCs) have been studied well in the prognosis for malignant diseases as liquid biopsy, but their contribution to tumor metastasis is not clearly defined. Here we report that CTCs could promote the metastatic colonization of disseminated carcinoma cells by inducing systemic inflammation and neutrophil recruitment to pre-metastatic organs. Depletion of neutrophils in vivo could effectively abrogate the promoting effect of CTCs on tumor cell metastasis. In the presence of CTCs, the pro-tumor function of neutrophils was augmented, whereas the antitumor function of neutrophils was suppressed. Mechanically, CTC-derived ligands for TLR2 and TLR4 (TLR2/4) induced the systemic inflammation, thus increasing the production of proinflammatory cytokines such as G-CSF and IL-6 that could induce the conversion of neutrophil function from tumor-suppressing to tumor-promoting. Moreover, CTCs induced the production of endogenous TLR2/4 ligands such as S100A8, S100A9, and SAA3, which may amplify the stimulating effect that induces the expression of proinflammatory cytokines. The promoting effect of CTCs on tumor cell metastasis could be abrogated by suppressing inflammatory response with IL-37, an anti-inflammatory cytokine, or blocking CTC-derived ligands for TLR2/4. Identification of the metastatic axis of CTCs/systemic inflammation/neutrophils may provide potential targets for preventing tumor cell metastasis.

Tumor-associated Lymphatic Endothelial Cells Promote Lymphatic Metastasis By Highly Expressing and Secreting SEMA4C.

PURPOSE: Lymphatic vessels are mainly regarded as passive conduits for the dissemination of cancer cells. In this study, we investigate whether and how the tumor-associated lymphatic vessels may play an active role in tumor metastasis. EXPERIMENTAL DESIGN: In situ laser capture microdissection of lymphatic vessels followed by cDNA microarray analysis was used to determine the expression profiling of lymphatic endothelial cells (LEC). Gene expression levels and activity of signaling pathways were measured by real-time RT-PCR, ELISA, or immunoblotting. Lymphangiogenesis was assessed by IHC. Lymph node metastasis was measured using fluorescence imaging. The effects of SEMA4C on lymphangiogenesis in vitro were evaluated using migration assay and tube-formation assay of LECs. RESULTS: Tumor-associated LECs are molecularly and functionally different from their normal counterparts. In addition to expressing high levels of membrane-bound SEMA4C, tumor-associated LECs also produced soluble SEMA4C (sSEMA4C). Increased serum sSEMA4C was detected in patients with breast cancer and cervical cancer. Patients with metastasis had much higher levels of serum sSEMA4C. sSEMA4C promoted lymphangiogenesis by activating PlexinB2-ERBB2 signaling in LECs, and promoted the proliferation and migration of tumor cells by activating PlexinB2-MET signaling, thus promoting lymphatic metastasis. Although the SEMA4C signaling pathways differ between LECs and tumor cells, RHOA activation was necessary for the effects of SEMA4C in both types of cells. CONCLUSIONS: Tumor-associated LECs produce sSEMA4C to promote lymphatic metastasis of tumors. Our results suggest that SEMA4C and RHOA might be potential therapeutic targets, and that higher serum sSEMA4C could be a marker for breast cancer and cervical cancer. Clin Cancer Res; 23(1); 214-24. ©2016 AACR.

14,15-EET induces the infiltration and tumor-promoting function of neutrophils to trigger the growth of minimal dormant metastases.

Infiltrating neutrophils are known to promote in the development of tumor. However, it is unclear whether and how neutrophils are involved in triggering the growth of dormant metastases. Here we show that 14,15-epoxyeicosatrienoic acid (14,15-EET) can trigger the growth of dormant micrometastases by inducing neutrophilic infiltration and converting neutrophil function. 14,15-EET triggered neutrophil infiltration in metastatic lesions by activating STAT3 and JNK pathways to induce the expression of human IL-8 and murine CXCL15 in corresponding tumor cells. The continuous expression of hIL-8/mCXCL15 was maintained by the sustained and enhanced activation of JNK pathway. 14,15-EET up-regulated miR-155 expression by activating STAT3 and JNK pathways. miR-155 in turn down-regulated the expression of SHIP1 and DET1, thus augmenting the activation of JNK and c-Jun. Moreover, the function of neutrophils was converted from tumor-suppressing to tumor-promoting by 14,15-EET in vivo. By inducing the production of G-CSF/IL-6 in vivo, 14,15-EET induced the enhancement of STAT3 activation in neutrophils to increase MMP-9 expression and decrease TRAIL expression. Neutrophil-derived MMP-9 was required for 14,15-EET to induce angiogenesis during the growth of dormant micrometastases. Depleting neutrophils or inhibiting hIL-8/mCXCL15 up-regulation resulted in the failure of 14,15-EET to promote the development of micrometastases. These findings reveal a mechanism through which the infiltration and tumor-promoting function of neutrophils could be induced to trigger the growth of dormant metastases, which might be a driving force for the tumor recurrence based on dormant metastases.

Sirtuin 6 promotes transforming growth factor-ß1/H2O2/HOCl-mediated enhancement of hepatocellular carcinoma cell tumorigenicity by suppressing cellular senescence.

Sirtuin 6 (SIRT6) can function as a tumor suppressor by suppressing aerobic glycolysis and apoptosis resistance. However, the negative effect of SIRT6 on cellular senescence implies that it may also have the potential to promote tumor development. Here we report that the upregulation of SIRT6 expression was required for transforming growth factor (TGF)-ß1 and H2O2/HOCl reactive oxygen species (ROS) to promote the tumorigenicity of hepatocellular carcinoma (HCC) cells. Transforming growth factor-ß1/H2O2/HOCl could upregulate SIRT6 expression in HCC cells by inducing the sustained activation of ERK and Smad pathways. Sirtuin 6 in turn abrogated the inducing effect of TGF-ß1/H2O2/HOCl on cellular senescence of HCC cells, and was required for the ERK pathway to efficiently suppress the expression of p16 and p21. Sirtuin 6 altered the effect of Smad and p38 MAPK pathways on cellular senescence, and contributed to the inhibitory effect of the ERK pathway on cellular senescence. However, SIRT6 was inefficient in antagonizing the promoting effect of TGF-ß1/H2O2 HOCl on aerobic glycolysis and anoikis resistance. Intriguingly, if SIRT6 expression was inhibited, the promoting effect of TGF-ß1/H2O2/HOCl on aerobic glycolysis and anoikis resistance was not sufficient to enhance the tumorigenicity of HCC cells. Suppressing the upregulation of SIRT6 enabled TGF-ß1/H2O2/HOCl to induce cellular senescence, thereby abrogating the enhancement of HCC cell tumorigenicity by TGF-ß1/H2O2/HOCl. These results suggest that SIRT6 is required for TGF-ß1/H2O2/HOCl to enhance the tumorigenicity of HCC cells, and that targeting the ERK pathway to suppress the upregulation of SIRT6 might be a potential approach in comprehensive strategies for the therapy of HCC.

Cyclin D1b splice variant promotes αvß3-mediated adhesion and invasive migration of breast cancer cells.

Cyclin D1b, a splice variant of the cell cycle regulator cyclin D1, holds oncogenic functions in human cancer. However, the mechanisms underlying cyclin D1b function remain poorly understood. Here we introduced wild-type cyclin D1a or cyclin D1b variant into non-metastatic MCF-7 cells. Our results show that ectopic expression of cyclin D1b promotes invasiveness of the cancer cells in a cyclin D1a independent manner. Specifically, cyclin D1b is found to modulate the expression of αvß3, which characterizes the metastatic phenotype, and enhance tumor cell invasive potential in cooperating with HoxD3. Notably, cyclin D1b promotes αvß3-mediated adhesion and invasive migration, which are associated with invasive potential of breast cancer cells. Further exploration indicates that cyclin D1b makes breast cancer cells more sensitive to toll-like receptor 4 ligand released from damaged tumor cells. These findings reveal a role of cyclin D1b as a possible mediator of αvß3 transcription to promote tumor metastasis.

SIX1 promotes tumor lymphangiogenesis by coordinating TGFß signals that increase expression of VEGF-C.

Lymphatic vessels are one of the major routes for the dissemination of cancer cells. Malignant tumors release growth factors such as VEGF-C to induce lymphangiogenesis, thereby promoting lymph node metastasis. Here, we report that sine oculis homeobox homolog 1 (SIX1), expressed in tumor cells, can promote tumor lymphangiogenesis and lymph node metastasis by coordinating with TGFß to increase the expression of VEGF-C. Lymphangiogenesis and lymph node metastasis in cervical cancer were closely correlated with higher expression of SIX1 in tumor cells. By enhancing VEGF-C expression in tumor cells, SIX1 could augment the promoting effect of tumor cells on the migration and tube formation of lymphatic endothelial cells (LEC) in vitro and lymphangiogenesis in vivo. SIX1 enhanced TGFß-induced activation of SMAD2/3 and coordinated with the SMAD pathway to modulate VEGF-C expression. Together, SIX1 and TGFß induced much higher expression of VEGF-C in tumor cells than each of them alone. Despite its effect in promoting VEGF-C expression, TGFß could inhibit lymphangiogenesis by directly inhibiting tube formation by LECs. However, the increased production of VEGF-C not only directly promoted migration and tube formation of LECs but also thwarted the inhibitory effect of TGFß on LECs. That is, tumor cells that expressed high levels of SIX1 could promote lymphangiogenesis and counteract the negative effects of TGFß on lymphangiogenesis by increasing the expression of VEGF-C. These findings provide new insights into tumor lymphangiogenesis and the various roles of TGFß signaling in tumor regulation. Our results also suggest that SIX1/TGFß might be a potential therapeutic target for preventing lymph node metastasis of tumor.

Neutrophils with protumor potential could efficiently suppress tumor growth after cytokine priming and in presence of normal NK cells.

In tumor-bearing state, the function of neutrophils is converted from tumor-suppressing to tumor-promoting. Here we report that priming with IFN-γ and TNF-α could convert the potential of neutrophils from tumor-promoting to tumor-suppressing. The neutrophils with protumor potential have not lost their responsiveness to IFN-γ and TNF-α. After priming with IFN-γ and TNF-α, the potential of the neutrophils to express Bv8 and Mmp9 genes was reduced. Conversely, the tumor-promotional neutrophils recovered the expression of Rab27a and Trail, resumed the activation levels of PI3K and p38 MAPK pathways in response to stimuli, and expressed higher levels of IL-18 and NK-activating ligands such as RAE-1, MULT-1, and H60. Therefore, the anti-tumor function of the neutrophils was augmented, including the cytotoxicity to tumor cells, the capability of degranulation, and the capacity to activate NK cells. Since the function of NK cells is impaired in tumor-bearing state, the administration of normal NK cells could significantly augment the efficiency of tumor therapy based on neutrophil priming. These findings highlight the reversibility of neutrophil function in tumor-bearing state, and suggest that neutrophil priming by IFN-γ/TNF-α might be a potential approach to eliminate residual tumor cells in comprehensive strategy for tumor therapy.

Adeno-associated virus-mediated expression of recombinant CBD-HepII polypeptide of human fibronectin inhibits metastasis of breast cancer.

CH50, a recombinant CBD-HepII polypeptide of human fibronectin, was shown to suppress tumor metastasis in murine hepatocarcinoma and melanoma models. However, the effect of CH50 on human cancer cells is still not clear. Here we evaluated the efficiency of CH50 delivered by recombinant adeno-associated virus (rAAV) vector for breast cancer treatment. Infection of the two human breast cancer cell line MDA-MB-231 and MDA-MB-468 with a rAAV2 vector encoding CH50 resulted in secretion of soluble CH50. In vitro rAAV-CH50 transduction inhibited adhesion to ECM molecules, and transwell migration and invasion of breast cancer cells induced by fibronectin. In both breast cancer cells, rAAV-CH50 targeted αVß3 signaling, namely inhibited the expression of αVß3, the activation of FAK, the upregulation of cdc2, and the production and activation of MMP-9 by ECM molecules stimulation. rAAV-mediated tail vein transfusion and stable expression of CH50 in the liver resulted in the long-term presence of CH50 in sera of nude mice. Sustained CH50 expression mediated by rAAV vector suppressed the growth and spontaneous metastasis of orthotopic breast cancer xenograft, experimental metastasis of circulating breast cancer cells, and improved the long-term survival of breast tumor-bearing mice. These findings suggest for the first time that rAAV-CH50 gene therapy may present a novel and promising strategy for treatment against metastatic breast cancer.

TLR4 ligand/H2O2 enhances TGF-ß1 signaling to induce metastatic potential of non-invasive breast cancer cells by activating non-Smad pathways.

TGF-ß1 has the potential to activate multiple signaling pathways required for inducing metastatic potential of tumor cells. However, TGF-ß1 was inefficient in inducing metastatic potential of many non-invasive human tumor cells. Here we report that the enhancement of TGF-ß1 signaling is required for inducing metastatic potential of non-invasive breast cancer cells. TGF-ß1 alone could not efficiently induce the sustained activation of Smad and non-Smad pathways in non-invasive breast cancer cells. TLR4 ligand (LPS) and H2O2 cooperated with TGF-ß1 to enhance the sustained activation of non-Smad pathways, including p38MAPK, ERK, JNK, PI3K, and NF-κB. The activation of MAPK and PI3K pathways resulted in a positive feed-back effect on TGF-ß1 signaling by down-regulating Nm23-H1 expression and up-regulating the expression of TßRI and TßRII, favoring further activation of multiple signaling pathways. Moreover, the enhanced TGF-ß1 signaling induced higher expression of SNAI2, which also promoted TßRII expression. Therefore, the sustained activation levels of both Smad and non-Smad pathways were gradually increased after prolonged stimulation with TGF-ß1/H2O2/LPS. Consistent with the activation pattern of signaling pathways, the invasive capacity and anoikis-resistance of non-invasive breast cancer cells were gradually increased after prolonged stimulation with TGF-ß1/H2O2/LPS. The metastatic potential induced by TGF-ß1/H2O2/LPS was sufficient for tumor cells to extravasate and form metastatic foci in an experimental metastasis model in nude mice. The findings in this study suggested that the enhanced signaling is required for inducing higher metastatic capacity of tumor cells, and that targeting one of stimuli or signaling pathways might be potential approach in comprehensive strategy for tumor therapy.

IL-6 cooperates with G-CSF to induce protumor function of neutrophils in bone marrow by enhancing STAT3 activation.

Neutrophils are known to have antitumor potential. However, in recent years the tumor-promoting effect of neutrophils has been well demonstrated. So far, it remains unclear what causes the conversion of neutrophil function from tumor suppressive to tumor promoting. In this article, we report that the conversion of murine neutrophil function occurs in bone marrow, and that IL-6 cooperation with G-CSF is required for this conversion. IL-6 cooperated with G-CSF to modulate neutrophils in bone marrow, altering the activation potential of signaling pathways in neutrophils, especially that of STAT3. Costimulation with G-CSF and IL-6 induced a higher level of phospho-STAT3 in neutrophils, which was further increased by upregulation of STAT3 expression in neutrophils owing to downregulation of IFN-ß expression in bone marrow macrophages by IL-6. Augmented STAT3 activation was crucial for upregulating the expression of Mmp9 and Bv8 genes and downregulating the expression of Trail and Rab27a genes in neutrophils. Moreover, G-CSF/IL-6-modulated neutrophils could not efficiently release azurophilic granules because of downregulation of Rab27a and inefficient activation of PI3K and p38 MAPK pathways. Because of premodulation by G-CSF and IL-6, neutrophils in response to complex stimuli in tumor released much less myeloperoxidase, neutrophil elastase, and TRAIL, but showed much higher expression of Mmp9 and Bv8 genes. Taken together, these results demonstrate that G-CSF and IL-6, despite their well-known physiological functions, could modulate the activation potential of signaling pathways in neutrophils, resulting in the production or release of the above-mentioned factors in a way that favors tumor angiogenesis and tumor growth.

Delivery of chemotherapeutic drugs in tumour cell-derived microparticles.

Cellular microparticles are vesicular plasma membrane fragments with a diameter of 100-1,000 nanometres that are shed by cells in response to various physiological and artificial stimuli. Here we demonstrate that tumour cell-derived microparticles can be used as vectors to deliver chemotherapeutic drugs. We show that tumour cells incubated with chemotherapeutic drugs package these drugs into microparticles, which can be collected and used to effectively kill tumour cells in murine tumour models without typical side effects. We describe several mechanisms involved in this process, including uptake of drug-containing microparticles by tumour cells, synthesis of additional drug-packaging microparticles by these cells that contribute to the cytotoxic effect and the inhibition of drug efflux from tumour cells. This study highlights a novel drug delivery strategy with potential clinical application.