Delayed apoptosis of tumor associated neutrophils in the absence of endogenous IFN‐β

The importance of neutrophils in tumor immune surveillance, invasive growth and angiogenesis becomes increasingly clear. Many of neutrophil activities are controlled by endogenous IFN‐β. Here, we provide evidence that endogenous IFN‐β is regulating the apoptosis of pro‐angiogenic tumor infiltrating neutrophils by influencing both, the extrinsic as well as the intrinsic apoptosis pathways. Accordingly, the life span of tumor associated neutrophils (TANs) is remarkably prolonged in tumor bearing Ifnb1^?/? mice compared to wild type controls. Lower expression of Fas, reactive oxygen species, active Caspase 3 and 9, as well as a change in expression pattern of proapoptotic and antiapoptotic members of the Bcl‐2 family and the major apoptosome constituent Apaf‐1 is observed under such conditions. In line with inhibition of apoptosis and the prolonged neutrophil survival, in the absence of endogenous IFN‐β, a strong enhancement of G‐CSF expression and PI3 Kinase phosphorylation is detected. These data explain the increased longevity of tumor infiltrating neutrophils and the accumulation of such cells in tumors. Taken together, our findings add to the important role of Type I IFN in immune surveillance against cancer.     

Breast cancer‐derived transforming growth factor‐β and tumor necrosis factor‐α compromise interferon‐α production by tumor‐associated plasmacytoid dendritic cells

We previously reported that plasmacytoid dendritic cells (pDCs) infiltrating breast tumors are impaired for their interferon‐α (IFN‐α) production, resulting in local regulatory T cells amplification. We designed our study to decipher molecular mechanisms of such functional defect of tumor‐associated pDC (TApDC) in breast cancer. We demonstrate that besides IFN‐α, the production by Toll‐like receptor (TLR)‐activated healthy pDC of IFN‐β and TNF‐α but not IP‐10/CXCL10 nor MIP1‐α/CCL3 is impaired by the breast tumor environment. Importantly, we identified TGF‐β and TNF‐α as major soluble factors involved in TApDC functional alteration. Indeed, recombinant TGF‐β1 and TNF‐α synergistically blocked IFN‐α production of TLR‐activated pDC, and neutralization of TGF‐β and TNF‐α in tumor‐derived supernatants restored pDCs' IFN‐α production. The involvment of tumor‐derived TGF‐β was further confirmed in situ by the detection of phosphorylated Smad2 in the nuclei of TApDC in breast tumor tissues. Mechanisms of type I IFN inhibition did not involve TLR downregulation but the inhibition of IRF‐7 expression and nuclear translocation in pDC after their exposure to tumor‐derived supernatants or recombinant TGF‐β1 and TNF‐α. Our findings indicate that targeting TApDC to restore their IFN‐α production might be an achievable strategy to induce antitumor immunity in breast cancer by combining TLR7/9‐based immunotherapy with TGF‐β and TNF‐α antagonists.     

Neutrophils recruit regulatory T‐cells into tumors via secretion of CCL17—A new mechanism of impaired antitumor immunity

The mechanisms by which tumor‐associated neutrophils (TANs) affect tumor growth are to a large extent unknown. Regulatory T‐cells (T‐regs) are functionally immune‐suppressive subsets of T‐cells. Depletion or inhibition of T‐regs can enhance antitumor immunity. We demonstrated both by RT‐PCR and by ELISA that murine TANs secrete significant amounts of the T‐regs chemoattractant, CCL17, much more than circulating or splenic neutrophils, and at a level progressively increasing during tumor development. Migration assays, both in vitro and in vivo, showed recruitment of T‐regs by TANs, which was inhibited with anti‐CCL17 monoclonal antibodies. Systemic neutrophil depletion in tumor‐bearing mice using anti‐Ly6G monoclonal antibodies reduced the migration of T‐regs into the tumors. We further showed, using flow cytometry, that CCL17 secretion by TANs is not limited to mouse models of cancer but is also relevant to human TANs. Our results suggest a new indirect mechanism by which TANs may inhibit antitumor immune activity, thus promoting tumor growth. We further describe, for the first time, a clear link between TANs and T‐regs acting together to impair antitumor immunity.     

Novel pegylated interferon‐β as strong suppressor of the malignant ascites in a peritoneal metastasis model of human cancer

Malignant ascites manifests as an end‐stage event during the progression of a number of cancers and lacks a generally accepted standard therapy. Interferon‐β (IFN‐β) has been used to treat several cancer indications; however, little is known about the efficacy of IFN‐β on malignant ascites. In the present study, we report on the development of a novel, engineered form of human and murine IFN‐β, each conjugated with a polyethylene glycol molecule (PEG‐hIFN‐β and PEG‐mIFN‐β, respectively). We provide evidence that these IFN‐β molecules retain anti‐viral potency comparable to unmodified IFN‐β in vitro and manifested improved pharmacokinetics in vivo. Interestingly, PEG‐mIFN‐β significantly inhibited the accumulation of ascites fluid and vascular permeability of the peritoneal membrane in models of ovarian cancer and gastric cancer cell xenograft mice. We further show that PEG‐hIFN‐β directly suppresses VEGF₁₆₅‐induced hyperpermeability in a monolayer of human vascular endothelial cells and that PEG‐mIFN‐β enhanced gene expression for a number of cell adhesion related molecules in mouse vascular endothelial cells. Taken together, these findings unveil a hitherto unrecognized potential of IFN‐β in maintaining vascular integrity, and provide proof‐of‐mechanism for a novel and long‐acting pegylated hIFN‐β for the therapeutic treatment of malignant ascites.     

Blockade of TGF‐β enhances tumor vaccine efficacy mediated by CD8+ T cells

Though TGF‐β inhibition enhances antitumor immunity mediated by CD8⁺ T cells in several tumor models, it is not always sufficient for rejection of tumors. In this study, to maximize the antitumor effect of TGF‐β blockade, we tested the effect of anti‐TGF‐β combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significantly delayed tumor growth, whereas anti‐TGF‐β antibodies alone did not show any antitumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti‐TGF‐β antibodies compared to vaccinated mice without anti‐TGF‐β, suggesting that anti‐TGF‐β synergistically enhanced irradiated tumor vaccine efficacy. CD8⁺ T‐cell depletion completely abrogated the vaccine efficacy, and so protection required CD8⁺ T cells. Depletion of CD25⁺ T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti‐TGF‐β did not change the number of CD25⁺ T regulatory cells in unvaccinated and vaccinated mice. Though the abrogation of CD1d‐restricted NKT cells, which have been reported to induce TGF‐β production by MDSC through an IL‐13‐IL‐4R‐STAT6 pathway, partially enhanced antitumor immunity regardless of vaccination, abrogation of the NKT cell‐IL‐13‐IL‐4R‐STAT‐6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti‐TGF‐β enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF‐β levels found in patients with cancer and that the effect is not dependent on TGF‐β solely from CD4⁺CD25⁺ T regulatory cells or the NKT cell‐IL‐13‐IL‐4R‐STAT‐6 immunoregulatory pathway.     

Interleukin‐10 gene transfer activates interferon‐γ and the interferon‐γ‐inducible genes Gbp‐1/Mag‐1 and Mig‐1 in mammary tumors

Expression of IL‐10 as a transgene inhibits murine mammary tumor growth and metastasis. Using differential display methodology, we sought genes whose expression was modulated by IL‐10. We compared mRNA isolated from parental murine mammary 66.1 tumors, as well as tumors derived from neo^r‐transfected cells and 6 different IL‐10‐expressing cell lines. We identified 2 cDNA products that were up‐regulated in all 6 IL‐10‐expressing tumors in comparison to parental and 66‐neo tumors. One cDNA corresponds to the murine guanylate‐binding protein gene Gbp‐1/Mag‐1. The other cDNA corresponds to the chemokine Mig‐1 (monokine induced by IFN‐γ). Both genes were originally identified in IFN‐γ‐activated macrophages or macrophage cell lines. We now report that cultured mammary epithelial tumor cell lines also express both genes in response to treatment with IFN‐γ and LPS. Furthermore, IFN‐γ mRNA is elevated in IL‐10‐expressing tumors in comparison with parental or neo‐transfected tumors. Thus, high‐level expression of IL‐10 as a transgene results in activation rather than suppression of IFN‐γ as well as 2 IFN‐γ‐inducible genes. Up‐regulation of host IFN‐γ is critical to anti‐tumor activity since IL‐10 no longer inhibits tumor growth in hosts with a deletion in the IFN‐γ gene. Additionally, Gbp‐1/Mag‐1 and Mig‐1 gene induction no longer occur in IFN‐γ mutant mice. Int. J. Cancer 80:624–629, 1999. © 1999 Wiley‐Liss, Inc.     

Cancer therapy with major histocompatibility complex‐deficient and interferon β‐producing myeloid cells derived from allogeneic embryonic stem cells

We previously established a method to generate myeloid cells with a proliferative capability from pluripotent stem cells and designated them iPS‐ML. Human iPS‐ML cells share features with physiological macrophages including the capability to infiltrate into cancer tissues. We observed therapeutic effects of human iPS‐ML cells expressing interferon β (iPS‐ML/interferon (IFN)‐β) in xenograft cancer models. However, assessment of host immune system‐mediated therapeutic and adverse effects of this therapy is impossible by xenograft models. We currently evaluated the therapeutic effects of a mouse equivalent of human iPS‐ML/IFN, a mouse embryonic stem (ES) cell‐derived myeloid cell line producing IFN (ES‐ML/IFN). The ES‐MLs producing IFN‐β (β‐ML) and IFN‐γ (γ‐ML) and originating from E14 ES cells derived from the 129 mouse strain (H‐2^b) were generated, and the MHC (H‐2K^b, D^b, and I‐A^b) genes of the ES‐ML/IFN were disrupted using the clustered regularly interspaced short palindromic repeats (CRISPR)/CAS9 method. We used the ES‐ML/IFN to treat allogeneic BALB/c mice (H‐2^d) transplanted with Colon26 cancer cells. Treatment with β‐ML but not with γ‐ML cells repressed the growth of colon cancer in the peritoneal cavity and liver. The transferred ES‐ML/IFN infiltrated into cancer tissues and enhanced infiltration of T cells into cancer tissues. ES‐ML/IFN therapy increased the number of immune cells in the lymphoid organs. Sensitization of both cancer antigen‐specific CD8⁺ T cells and natural killer (NK) cells were enhanced by the therapy, and CD8⁺ T cells were essential for the therapeutic effect, implying that donor MHC‐deficient β‐ML exhibited a therapeutic effect through the activation of host immune cells derived from allogeneic recipient mice. The results suggested the usefulness of HLA‐deficient human iPS‐ML/IFN‐β cells for therapy of HLA‐mismatched allogeneic cancer patients.     

Correlation between CXCR4/CXCR7/CXCL12 chemokine axis expression and prognosis in lymph‐node‐positive lung cancer patients

The CXCR4/CXCR7/CXCL12 chemokine axis plays important roles in the migration of tumor cells during cancer development by modulating site‐specific distant metastasis including to regional lymph nodes. We investigated the correlation of these chemokine expressions to prognosis in lymph‐node‐positive non‐small‐cell lung cancer (NSCLC) patients. A total of 140 surgically resected specimens of primary site (PS) and metastatic lymph nodes (MLN) of NSCLC involving hilar and/or mediastinal lymph nodes (N1‐2) were collected. CXCR4, CXCR7 and CXCL12 expressions were evaluated. Cox regression analysis was performed to determine whether these chemokines were independent prognostic factors in N1‐2 NSCLC. High expression of CXCR4 in PS and CXCL12 in MLN was associated with poor overall survival (OS) (P = .025 and .033, respectively). Significant correlations between CXCR4 expression in PS and CXCL12 expression in MLN were observed (P = .040). There was significant difference in OS between 2 groups according to expressions of CXCR4 in PS and CXCL12 in MLN (P = .0033). Expression of CXCL12 in MLN was identified as an independent prognostic factor (HR 1.79, 95% CI 1.08‐3.04, P = .023). CXCL12 in MLN was mainly expressed by tumor cells compared with stromal cells (56% vs 25%, respectively, P < .0001). CXCR4/CXCL12 may play roles in tumor progression in MLN and is associated with poor prognosis of lymph‐node‐positive NSCLC patients.     

Perivascular expression of CXCL9 and CXCL12 in primary central nervous system lymphoma: T‐cell infiltration and positioning of malignant B cells

Primary central nervous system lymphomas (PCNSL) are aggressive malignancies confined to the CNS, mostly of diffuse large B‐cell histotype. Despite improved understanding of the malignant B cells, little is known on the tumor microenvironment and on the response of the adaptive immunity against PCNSL. We investigated the phenotype of tumor infiltrating lymphocytes (TILs), and the expression of chemokines that could affect malignant B cells and trafficking of TILs. TILs and chemokine expression were evaluated by immunohistochemistry and in situ hybridization. Furthermore, we performed in vitro migration assays to analyze the migratory capacity of lymphocytes and malignant B cells toward chemokines and chemokine heterocomplexes. We show in 22 cases of PCNSL from immunocompetent patients that CD8⁺ T cells represent the majority of TILs in the tumor mass. They tend to accumulate in perivascular areas, show Granzyme B expression and proliferate in situ. Their localization and density correlates with the expression of the inflammatory chemokine CXCL9, which is transcribed and translated by perivascular macrophages and pericytes in the perivascular microenvironment. Moreover, CXCL9 and CXCL12 are coexpressed on the tumor vasculature and form heterocomplexes. In the presence of CXCL9, CXCL12‐induced migration is enhanced not only on CXCR4⁺/CXCR3⁺/CD8⁺ T cells but also on CXCR4⁺/CXCR3^? malignant B cells. These findings indicate the presence of a strong chemoattractant stimulus in the perivascular microenvironment, which might serve as regulator for the recruitment of TILs and for the angiocentric positioning of malignant B cells in the perivascular cuff.     

Small interfering RNA‐mediated CXCR1 or CXCR2 knock‐down inhibits melanoma tumor growth and invasion

CXCR1 and CXCR2 are receptors for CXCL‐8 and are differentially expressed on melanoma and endothelial cells. In this study, we determined the functional role of these receptors in melanoma progression. We stably knock‐down the expression of CXCR1 and/or CXCR2 in A375‐SM (SM; high metastatic) human melanoma cells by short‐hairpin RNA transfection. Cell proliferation, migration, invasion, ERK phosphorlyation and cytoskeletal rearrangements were carried out in vitro. In vivo growth was evaluated using murine subcutaneous xenograft model. Our data demonstrate that knock‐down of CXCR1 and/or CXCR2 expression, inhibited melanoma cell proliferation, survival, migration and invasive potential in vitro. Moreover, we also observed inhibition of ERK phosphorylation and cytoskeltal rearrangement in SM‐shCXCR1, SM‐shCXCR2 and SM‐shCXCR1/2 cells. Furthermore, when SM‐shCXCR1 or SM‐shCXCR2 cells implanted in nude mice, tumor growth, proliferation and microvessel density was significantly inhibited as compared to SM‐control cells. In addition, we observed a significant increase in melanoma cell apoptosis in SM‐shCXCR1 and SM‐shCXCR2 tumors compared to SM‐control tumors. Together, these data demonstrate that CXCR1 and CXCR2 expression play a critical role in human melanoma tumor progression and, functional blockade of CXCR1 and CXCR2 could be potentially used for future therapeutic intervention in malignant melanoma.     

Host STAT2/type I interferon axis controls tumor growth

The role of STAT2 in mediating the antigrowth effects of type I interferon (IFN) is well‐documented in vitro. Yet evidence of IFN‐activated STAT2 as having tumor suppressor function in vivo and participation in antitumor immunity is lacking. Here we show in a syngeneic tumor transplantation model that STAT2 reduces tumor growth. Stat2^?/? mice formed larger tumors compared to wild type (WT) mice. IFN‐β treatment of Stat2^?/? mice did not cause tumor regression. Gene expression analysis revealed a small subset of immunomodulatory genes to be downregulated in tumors established in Stat2^?/? mice. Additionally, we found tumor antigen cross‐presentation by Stat2^?/? dendritic cells to T cells to be impaired. Adoptive transfer of tumor antigen specific CD8⁺ T cells primed by Stat2^?/? dendritic cells into tumor‐bearing Stat2^?/? mice did not induce tumor regression with IFN‐β intervention. We observed that an increase in the number of CD4⁺ and CD8⁺ T cells in the draining lymph nodes of IFN‐β‐treated tumor‐bearing WT mice was absent in IFN‐β treated Stat2^?/? mice. Thus our study provides evidence for further evaluation of STAT2 function in cancer patients receiving type I IFN based immunotherapy.     

Peptide inhibitors of transforming growth factor‐β enhance the efficacy of antitumor immunotherapy

Transforming growth factor‐β (TGF‐β) is a cytokine with potent immunosuppressive effects and is overexpressed in many tumors. Therefore, development of molecules able to inhibit TGF‐β is of paramount importance to improve the efficacy of antitumor immunotherapy. TGF‐β inhibitor peptides P144 and P17 were combined with the administration of adjuvant molecules poly(I:C) and agonistic anti‐CD40 antibodies, and their effect on the growth of E.G7‐OVA established tumors and on antitumor immune response was evaluated. Tumor rejection efficacy of a single administration of adjuvants was enhanced from 15 to 70 % when combined with repeated injections of TGF‐β inhibitor peptides. Simultaneous administration of adjuvants and TGF‐β inhibitor peptides was required for maximal therapeutic efficacy. Although tumor cells produced TGF‐β, it was found that the beneficial effect of peptide administration was mainly due to the inhibition of TGF‐β produced by regulatory CD4⁺CD25⁺ T cells rather than by tumor cells. The enhanced antitumor effect was accompanied by a higher activity of dendritic cells, natural killer cells and tumor antigen‐specific T cells, as well as by a decrease in the number of myeloid‐derived suppressor cells. In conclusion, administration of peptide inhibitors of TGF‐β in therapeutic vaccination enhances the efficacy of immunotherapy by increasing antitumor immune responses. These peptide inhibitors may have important applications for current immunotherapeutic strategies. © 2009 UICC     

The lack of type I interferon induces neutrophil‐mediated pre‐metastatic niche formation in the mouse lung

Metastases are the major cause of death from cancer. Thus, understanding the regulation of metastatic processes is of utmost importance. Here we show that mice with impaired type I IFN signaling (Ifnar1^‐/‐) develop more lung metastases in the 4T1 mammary and LLC lung carcinoma model, compared to control mice. In Ifnar1^‐/‐ mice, higher metastasis load is accompanied by massive neutrophil accumulation in lungs. Elevated G‐CSF levels in serum and enhanced CXCR2 expression on neutrophils are most likely responsible for this phenomenon. Lung infiltrating neutrophils facilitate an improved pre‐metastatic niche formation, supporting more efficient tumor cell extravasation and proliferation in this organ. This is due to the enhanced expression of pro‐metastatic proteins, like Bv8, MMP9, S100A8 and S100A9. Development of pre‐metastatic niche together with reduced neutrophil cytotoxicity against tumor cells results in enhanced metastatic processes in Ifnar1^‐/‐ mice. Overall, our findings describe a novel role for IFN during metastasis development and suggest that new treatment strategies should be considered for prevention of metastasis formation in patients.     

Cellular context‐dependent “colors” of transforming growth factor‐β signaling

Transforming growth factor (TGF)‐β signaling has interesting characteristics in the context of cancer. Although perturbations of TGF‐β signaling are strongly implicated in cancer progression, TGF‐β signaling has both tumor‐suppressive and tumor‐promoting effects. For example, TGF‐β inhibits cancer cell proliferation in some cellular contexts, but promotes it in others. Although several approaches to treating cancer have been considered using TGF‐β‐based therapeutic strategies, the contradictory behaviors of TGF‐β have made these approaches complex. To put them to practical use, either the tumor‐suppressive or tumor‐promoting arm needs to be specifically manipulated. However, there is virtually no method to specifically regulate a certain cell response induced by TGF‐β. In this review, we first consider the basic machinery of TGF‐β signaling, and describe several cell responses induced by TGF‐β stimulation in specific contexts. Mechanisms by which TGF‐β can induce several responses in a cellular context‐dependent fashion are discussed with established paradigms and models. We also address perspectives on the specific control of only a subset of numerous cell responses induced by TGF‐β stimulation. Such methods will aid specific regulation of either the tumor‐suppressive or tumor‐promoting arm of the TGF‐β pathway and in realization of TGF‐β‐based treatment of malignant tumors. (Cancer Sci 2010; 101: 306–312)     

TLR2/6 agonists and interferon‐gamma induce human melanoma cells to produce CXCL10

Clinical approaches to treat advanced melanoma include immune therapies, whose benefits depend on tumor‐reactive T‐cell infiltration of metastases. However, most tumors lack significant immune infiltration prior to therapy. Selected chemokines promote T‐cell migration into tumors; thus, agents that induce these chemokines in the tumor microenvironment (TME) may improve responses to systemic immune therapy. CXCL10 has been implicated as a critical chemokine supporting T‐cell infiltration into the TME. Here, we show that toll‐like receptor (TLR) agonists can induce chemokine production directly from melanoma cells when combined with IFNγ treatment. We find that TLR2 and TLR6 are widely expressed on human melanoma cells, and that TLR2/6 agonists (MALP‐2 or FSL‐1) synergize with interferon‐gamma (IFNγ) to induce production of CXCL10 from melanoma cells. Furthermore, melanoma cells and immune cells from surgical specimens also respond to TLR2/6 agonists and IFNγ by upregulating CXCL10 production, compared to treatment with either agent alone. Collectively, these data identify a novel mechanism for inducing CXCL10 production directly from melanoma cells, with TLR2/6 agonists +IFNγ and raise the possibility that intratumoral administration of these agents may improve immune signatures in melanoma and have value in combination with other immune therapies, by supporting T‐cell migration into melanoma metastases.     

3‐Methylcholanthrene‐induced transforming growth factor‐β‐producing carcinomas,but not sarcomas,are refractory to regulatory T cell‐depletion therapy

Regulatory T cells (Tregs) are major immunosuppressors in tumor‐bearing hosts. Although Treg‐depletion therapy has been shown to induce a complete cure in tumor‐bearing mice, this treatment is not always successful. Using 3‐methylcholanthrene‐induced primary mouse tumors, we examined the distinct regulation of Treg‐mediated immunosuppression between carcinomas and sarcomas. We showed that the number of Tregs was greatly increased in squamous cell carcinoma (SCC)‐bearing mice compared with sarcoma‐bearing mice. This appeared to be because SCC produced higher levels of active transforming growth factor (TGF)‐β, which is essential for inducing Tregs, compared with sarcoma. Moreover, SCC, but not sarcomas, were refractory to Treg‐depletion therapy by treatment with anti‐CD25 mAb. The refractoriness of SCC against Treg‐depletion therapy was due to the rapid recovery of Tregs in SCC‐bearing mice compared with sarcoma‐bearing mice. However, combination treatment of anti‐TGF‐β mAb with anti‐CD25 mAb caused a significant reduction in Treg recovery and induced a complete cure in SCC‐bearing mice. Thus, we showed the refractoriness of mouse carcinoma against Treg‐depletion therapy using anti‐CD25 mAb treatment. We also proposed a novel Treg‐blocking combination therapy using anti‐CD25 mAb and anti‐TGF‐β mAb to induce a complete cure of tumor‐bearing hosts. (Cancer Sci 2010; 101: 855–861)