Blockade of Myd88 signaling induces antitumor effects by skewing the immunosuppressive function of myeloid‐derived suppressor cells

Myd88 is an important adaptor molecule for the activation of NADPH oxidase and arginase‐1, which are responsible for the suppressive function of myeloid‐derived suppressor cells (MDSCs). When wild‐type and Myd88^?/? mice were subcutaneously injected with CT26 colon cancer cells expressing human Her‐2/neu, tumor growth was retarded in Myd88^?/? mice than in wild‐type mice. Although the generation of CD11b⁺Gr‐1⁺ MDSCs was less in Myd88^?/? mice than in wild‐type mice, Myd88^?/? mice having tumor masses still had significant quantities of MDSCs, suggesting that MDSC generation might be independent of Myd88 signaling. However, MDSCs obtained from tumor‐bearing Myd88^?/? mice failed to suppress antigen‐specific proliferation of CD8⁺ T cells and CD4⁺ T cells, whereas MDSCs from wild‐type mice significantly suppressed both types of T cells. Consistent with this, we found that the levels of costimulatory molecules and MHC class II were significantly increased in MDSCs obtained from Myd88^?/? mice compared with wild‐type mice after tumor challenge. Furthermore, CD4⁺ T cells residing in tumor‐draining lymph nodes of Myd88^?/? mice secreted more TNF‐α than those of wild‐type mice. Finally, the blockade of Myd88 signaling by treatment with Myd88 inhibitory peptide, during later tumor stages, significantly inhibited the growth of immunogenic tumors. Overall, these data suggest that signaling through the Myd88 adaptor molecule is critical for the direct suppressive function of MDSCs and approaches to block Myd88‐mediated signaling in MDSCs might be effective to inhibit the immunosuppressive function of MDSCs.     

Tumor‐specific cytotoxic T cell generation and dendritic cell function are differentially regulated by interleukin 27 during development of anti‐tumor immunity

Interleukin (IL‐) 27 is a member of IL‐12 cytokine family with Th1‐promoting and anti‐inflammatory effects. IL‐27 has been shown to facilitate tumor‐specific cytotoxic T lymphocyte (CTL) induction against various tumors. However, IL‐27 suppresses cytokine production of lymphocytes and antigen‐presenting function of dendritic cells (DCs). To examine the in vivo role of IL‐27 in generation of anti‐tumor immunity, we examined IL‐27‐mediated antitumor‐effects using WSX‐1 (IL‐27 receptor α chain)‐deficient (WSX‐1^?/?) mice. In WSX‐1^?/? mice inoculated with B16 melanoma cells, tumor growth was higher than in wild‐type (WT) mice. Accordingly, tumor‐specific CTL generation was lower in WSX‐1^?/? mice than in WT mice. CTL induction in WSX‐1^?/? mice was not restored by transfer of WT DCs pulsed with TRP2 peptide, indicating that IL‐27 is directly required for generation of tumor‐specific CTLs. However, when transferred into tumor‐bearing mice, WSX‐1^?/? DCs pulsed with TRP2 peptide was more potent than WT DCs in tumor growth inhibition and generation of CTLs, indicating suppressive effects of IL‐27 on DC function. Finally, the combination of WT CD8⁺ T cells and KO DCs is more potent in generation of antigen‐specific CTLs than any other combinations. Expression of perforin gene and percentages of tumor‐specific CD8⁺ T cells were also the highest in the combination of WT CD8+ T cells and WSX‐1^?/? DCs. It was thus revealed that IL‐27 promotes CTL generation while suppressing DC function during generation of tumor immunity. The combination of WT T cells and IL‐27 signal‐defective DCs may have therapeutic potential against tumors. © 2008 Wiley‐Liss, Inc.     

P‐glycoprotein (MDR1/ABCB1) restricts brain accumulation and cytochrome P450‐3A (CYP3A) limits oral availability of the novel ALK/ROS1 inhibitor lorlatinib

Lorlatinib (PF‐06463922) is a promising oral anaplastic lymphoma kinase (ALK) and ROS1 inhibitor currently in Phase III clinical trials for treatment of non‐small‐cell lung cancer (NSCLC) containing an ALK rearrangement. With therapy‐resistant brain metastases a major concern in NSCLC, lorlatinib was designed to have high membrane and blood–brain barrier permeability. We investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, and the multispecific drug‐metabolizing enzyme CYP3A in plasma pharmacokinetics and tissue distribution of lorlatinib using genetically modified mouse strains. In vitro, human ABCB1 and mouse Abcg2 modestly transported lorlatinib. Following oral lorlatinib administration (at 10 mg/kg), brain accumulation of lorlatinib, while relatively high in wild‐type mice, was still fourfold increased in Abcb1a/1b^?/? and Abcb1a/1b;Abcg2^?/? mice, but not in single Abcg2^?/? mice. Lorlatinib plasma levels were not altered. Oral coadministration of the ABCB1/ABCG2 inhibitor elacridar increased the brain accumulation of lorlatinib in wild‐type mice fourfold, that is, to the same level as in Abcb1a/1b;Abcg2^?/? mice, without altering plasma exposure. Similar results were obtained for lorlatinib testis accumulation. In Cyp3a^?/? mice, the plasma exposure of lorlatinib was increased 1.3‐fold, but was then twofold reduced upon transgenic overexpression of human CYP3A4 in liver and intestine, whereas relative tissue distribution of lorlatinib remained unaltered. Our data indicate that lorlatinib brain accumulation is substantially limited by P‐glycoprotein/ABCB1 in the blood–brain barrier, but this can be effectively reversed by elacridar coadministration. Moreover, oral availability of lorlatinib is markedly restricted by CYP3A4 activity. These insights may be used in optimizing the therapeutic application of lorlatinib.     

Increased oral availability and brain accumulation of the ALK inhibitor crizotinib by coadministration of the P‐glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) inhibitor elacridar

Crizotinib is an oral tyrosine kinase inhibitor approved for treating patients with non‐small cell lung cancer (NSCLC) containing an anaplastic lymphoma kinase (ALK) rearrangement. We used knockout mice to study the roles of P‐glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in plasma pharmacokinetics and brain accumulation of oral crizotinib, and the feasibility of improving crizotinib kinetics using coadministration of the dual ABCB1/ABCG2 inhibitor elacridar. In vitro, crizotinib was a good transport substrate of human ABCB1, but not of human ABCG2 or murine Abcg2. With low‐dose oral crizotinib (5 mg/kg), Abcb1a/1b^?/? and Abcb1a/1b;Abcg2^?/? mice had an approximately twofold higher plasma AUC than wild‐type mice, and a markedly (～40‐fold) higher brain accumulation at 24 hr. Also at 4 hr, crizotinib brain concentrations were ～25‐fold, and brain‐to‐plasma ratios ～14‐fold higher in Abcb1a/1b^?/? and Abcb1a/1b;Abcg2^?/? mice than in wild‐type mice. High‐dose oral crizotinib (50 mg/kg) resulted in comparable plasma pharmacokinetics between wild‐type and Abcb1a/1b^?/? mice, suggesting saturation of intestinal Abcb1. Nonetheless, brain accumulation at 24 hr was still ～70‐fold higher in Abcb1a/1b^?/? than in wild‐type mice. Importantly, oral elacridar coadministration increased the plasma and brain concentrations and brain‐to‐plasma ratios of crizotinib in wild‐type mice, equaling the levels in Abcb1a/1b;Abcg2^?/? mice. Our results indicate that crizotinib oral availability and brain accumulation were primarily restricted by Abcb1 at a non‐saturating dose, and that coadministration of elacridar with crizotinib could substantially increase crizotinib oral availability and delivery to the brain. This principle might be used to enhance therapeutic efficacy of crizotinib against brain metastases in NSCLC patients.     

CENP‐R acts bilaterally as a tumor suppressor and as an oncogene in the two‐stage skin carcinogenesis model

CENP‐R is a component of the CENP‐O complex, including CENP‐O, CENP‐P, CENP‐Q, CENP‐R, and CENP‐U and is constitutively localized to kinetochores throughout the cell cycle in vertebrates. CENP‐R‐deficient chicken DT40 cells are viable and show a very minor effect on mitosis. To investigate the functional roles of CENP‐R in vivo, we generated CENP‐R‐deficient mice (Cenp‐r^?/?). Mice heterozygous or homozygous for Cenp‐r null mutation are viable and healthy, with no apparent defect in growth and morphology, indicating Cenp‐r is not essential for normal development. Accordingly, to investigate the role of the Cenp‐r gene in skin carcinogenesis, we subjected Cenp‐r^?/? mice to the 7,12‐dimethylbenz(a)anthracene (DMBA)/TPA chemical carcinogenesis protocol and monitored tumor development. As a result, Cenp‐r^?/? mice initially developed significantly more papillomas than control wild‐type mice. However, papillomas in Cenp‐r^?/? mice showed a decrease of proliferative cells and an increase of apoptotic cells. As a result, they did not grow bigger and some papillomas showed substantial regression. Furthermore, papillomas in Cenp‐r^?/? mice showed lower frequency of malignant conversion to squamous cell carcinomas. These results indicate Cenp‐r functions bilaterally in cancer development: during early developmental stages, Cenp‐r functions as a tumor suppressor, but during the expansion and progression of papillomas it functions as a tumor‐promoting factor.     

Expression of TLR9 in tumor‐infiltrating mononuclear cells enhances angiogenesis and is associated with a worse survival in lung cancer

Toll‐like receptors (TLRs) play a crucial role in the innate and adaptive immune responses against microbial infection, tissue injury and cancer. Ligands of TLR9 have been developed as therapy in non‐small‐cell lung carcinoma (NSCLC). However, phase III clinical trials in metastatic NSCLC were negative. Our objective was to determine whether TLR9 affects tumor growth. We generated a mouse model of lung adenocarcinoma (ADC) mutated for K‐ras (K‐ras^LA1), with and without TLR9 inactivation (K‐ras^LA1TLR9^?/? and K‐ras^LA1TLR9^+/+, respectively). TLR9 was functionally expressed only in mononuclear cells of K‐ras^LA1TLR9^+/+ mice. These mice had significantly worse survival and a higher tumor burden than K‐ras^LA1TLR9^?/? mice. Lung tumors were analyzed for 24 cytokines/growth factors using Bio‐Plex multiplex bead‐based assays. Factor VIII was assessed by immunochemistry. Tumors from K‐ras^LA1TLR9^+/+ mice were characterized by an angiogenic phenotype with higher concentrations of vascular endothelial growth factor (VEGF) and higher microvessel density than from K‐ras^LA1TLR9^?/? mice. LKR13 cells, an ADC cell line derived from K‐ras^LA1 mice, were subcutaneously injected into TLR9^?/? and TLR9^+/+ mice. Syngeneic tumors regressed in TLR9^?/? but not in TLR9^+/+ mice. Peripheral blood mononuclear cells from TLR9^?/? mice released less VEGF than those from TLR9^+/+ mice. In 61 patients with early‐stage NSCLC, TLR9 was expressed in mononuclear cells that infiltrated tumors, as assessed by immunochemistry, and contributed to worse survival. Our results suggest that TLR9 expression in mononuclear cells was associated with an angiogenic phenotype and promoted lung cancer progression. These findings may aid clinical development of TLR9 ligands to treat cancers.     

MicroRNA‐155 deficiency enhances the recruitment and functions of myeloid‐derived suppressor cells in tumor microenvironment and promotes solid tumor growth

Immune cells in tumor microenvironment play a prominent role in tumor progression and metastasis. MicroRNA‐155 (miR‐155) represents an important player in innate and adaptive immunity by regulating differentiation, maturation and activation of macrophages, dendritic cells, B cells and T cells. However, the role of miR‐155 expression in immune cells in solid tumor development is less elucidated. Our current study showed that both B16‐F10 melanoma and Lewis lung carcinoma tumors grew much faster in bic/miR‐155 knockout (miR‐155^?/?) mice along with an increase of myeloid‐derived suppressor cells (MDSCs) accumulation in tumors, compared to that in wild‐type mice. Bone marrow transplantation study showed that bone marrow miR‐155 deficiency could replicate the above tumor‐promoting phenotype. In vitro study demonstrated that tumor‐infiltrating miR‐155^?/? MDSCs showed greater migration ability and expressed higher level of multiple chemokines. Furthermore, we found that the level of HIF‐1α, a direct target of miR‐155, was increased in miR‐155 deficient MDSCs, and that the increased HIF‐1α upregulated CXCL1, CXCL3 and CXCL8 expression in MDSCs, contributing to the enhanced recruitment of miR‐155^?/? MDSCs to the tumors. Moreover, miR‐155^?/? MDSCs showed enhanced immunosuppressive and pro‐angiogenic capacities. Taken together, our study, for the first time, demonstrated that miR‐155 deficiency promoted solid tumor growth through increasing the recruitment of MDSCs to tumor microenvironment and enhancing the tumor‐promoting functions of the recruited MDSCs. Thus, upregulating miR‐155 expression in MDSCs may be developed as a therapeutic approach to halt tumor development.     

Cooperation of tumor‐derived HBx mutants and p53‐249ser mutant in regulating cell proliferation,anchorage‐independent growth and aneuploidy in a telomerase‐immortalized normal human hepatocyte‐derived cell line

Hepatocellular carcinoma (HCC) is a common cancer, and hepatitis B virus (HBV) is a major etiological agent. Convincing epidemiological and experimental evidence also links HCC to aflatoxin, a naturally occurring mycotoxin that produces a signature p53‐249^ser mutation. Recently, we have reported that tumor‐derived HBx variants encoded by HBV exhibited attenuated transactivation and proapoptotic functions but retained their ability to block p53‐mediated apoptosis. These results indicate that mutations in HBx may contribute to the development of HCC. In this study, we determined whether tumor‐derived HBx mutants along, or in cooperation with p53‐249^ser, could alter cell proliferation and chromosome stability of normal human hepatocytes. To test this hypothesis, we established a telomerase immortalized normal human hepatocycte line HHT4 that exhibited a near diploid karyotype and expressed many hepatocyte‐specific genes. We found that overexpression one of the tumor‐derived HBx mutants, CT, significantly increased colony forming efficiency (CFE) while its corresponding wild‐type allele CNT significantly decreased CFE in HHT4 cells. p53‐249^ser rescued CNT‐mediated inhibition of colony formation. Although HHT4 cells lacked an anchorage independent growth capability as they did not form any colonies in soft agar, the CT‐expressing HHT4 cells could form colonies, which could be significantly enhanced by p53‐249^ser. Induction of aneuploidy could be observed in HHT4 cells expressing CT, but additionally recurring chromosome abnormalities could only be detected in cells coexpressing CT and p53‐249^ser. Our results are consistent with the hypothesis that certain mutations in HBx and p53 at codon 249 may cooperate in contributing to liver carcinogenesis.     

Blockade of MK2 is protective in inflammation‐associated colorectal cancer development

Chronic inflammation is a risk factor for colorectal cancer. The MAPK‐activated protein kinase 2 (MK2) pathway controls multiple cellular processes including p38‐dependent inflammation. This is the first study to investigate the role of MK2 in development of colitis‐associated colon cancer (CAC). Herein, we demonstrate that MK2^?/? mice are highly resistant to neoplasm development when exposed to AOM/DSS, while wild type (WT) C57BL/6 develop multiple neoplasms with the same treatment. MK2‐specific cytokines IL‐1, IL‐6 and TNF‐α were substantially decreased in AOM/DSS treated MK2^?/? mouse colon tissues compared with WT mice, which coincided with a marked decrease in macrophage influx. Restoring MK2‐competent macrophages by injecting WT bone marrow derived macrophages into MK2^?/? mice led to partial restoration of inflammatory cytokine production with AOM/DSS treatment; however, macrophages were not sufficient to induce neoplasm development. These results indicate that MK2 functions as an inflammatory regulator to promote colonic neoplasm development and may be a potential target for CAC.     

Modulatory effects of adiponectin on the polarization of tumor‐associated macrophages

The plasticity of macrophages with selective functional phenotypes partially arises in respective to their microenvironment. Tumor‐associated macrophages (TAMs) may promote disease progression with tumor specific manner. Here we report that in pediatric malignant soft‐tissue tumors, the presence of TAMs and expression of adiponectin (APN) are heterogeneous. Both APN and TAMs had high expression in rhabdomyosarcoma, especially in the malignant subtype, alveolar rhabdomyosarcoma. To investigate the mode of action of APN on TAM activation, a murine MN/MCA1 sarcoma model was used. The Results revealed that exogenous APN had no effect on MN/MCA1 proliferation but tumor size was markedly reduced in apn^?/? mice versus WT controls. The accumulation of TAMs in apn^?/? mice was also reduced which correlated to downregulated serum levels of MCP‐1. Likewise, TAMs in apn^?/? mice exhibited a M1‐like phenotype, characterized by increase in MHC II^high population and M1 phenotypic markers, such as iNOS gene and serum TNF‐α accompanied by a decrease in M2 markers, namely YM1 gene and serum IL‐10. In addition, APN deficiency increased the number of CD4⁺ T cells, CD8⁺ T cells and NK cells in tumors and reduced tumor metastasis. The altered phenotype of TAMs in apn^?/? mice was associated with a marked decrease in phospho‐p38 and treatment with a p38 MAPK inhibitor significantly reduced tumor size and increased MHC II expression on TAMs in WT mice, implying p38 MAPK signaling pathway may contribute to APN‐mediated TAM polarization. Collectively, our findings suggest that APN may have a potential role in regulating soft tissue sarcoma growth.     

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.     

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.     

A transplantable TH‐MYCN transgenic tumor model in C57Bl/6 mice for preclinical immunological studies in neuroblastoma

Current multimodal treatments for patients with neuroblastoma (NBL), including anti‐disialoganglioside (GD2) monoclonal antibody (mAb) based immunotherapy, result in a favorable outcome in around only half of the patients with advanced disease. To improve this, novel immunocombinational strategies need to be developed and tested in autologous preclinical NBL models. A genetically well‐explored autologous mouse model for NBL is the TH‐MYCN model. However, the immunobiology of the TH‐MYCN model remains largely unexplored. We developed a mouse model using a transplantable TH‐MYCN cell line in syngeneic C57Bl/6 mice and characterized the immunobiology of this model. In this report, we show the relevance and opportunities of this model to study immunotherapy for human NBL. Similar to human NBL cells, syngeneic TH‐MYCN‐derived 9464D cells endogenously express the tumor antigen GD2 and low levels of MHC Class I. The presence of the adaptive immune system had little or no influence on tumor growth, showing the low immunogenicity of the NBL cells. In contrast, depletion of NK1.1+ cells resulted in enhanced tumor outgrowth in both wild‐type and Rag1^?/? mice, showing an important role for NK cells in the natural anti‐NBL immune response. Analysis of the tumor infiltrating leukocytes ex vivo revealed the presence of both tumor associated myeloid cells and T regulatory cells, thus mimicking human NBL tumors. Finally, anti‐GD2 mAb mediated NBL therapy resulted in ADCC in vitro and delayed tumor outgrowth in vivo. We conclude that the transplantable TH‐MYCN model represents a relevant model for the development of novel immunocombinatorial approaches for NBL patients.     

Lymphotoxin‐β receptor activation by lymphotoxin‐α1β2 and LIGHT promotes tumor growth in an NFκB‐dependent manner

Lymphotoxin beta receptor (LTβR) activation on mouse fibrosarcoma cells (BFS‐1) results in enhanced solid tumor growth paralleled by increased angiogenesis induced by the expression of pro‐angiogenic CXCL2. In our study, we demonstrate that both functional ligands of the LTβR, namely LTα₁β₂ and LIGHT, are involved in the activation of LTβR in solid fibrosarcomas. To identify whether the lymphocyte population is involved in the activation of LTβR in these fibrosarcoma tumors, we used conditional LTβ‐deficient mice that specifically lack LTβ expression either on T cells (T‐LTβ^?/?) or on B cells (B‐LTβ^?/?). Solid tumor growth was reduced in both mouse strains when compared to tumor growth in wild‐type mice, indicating the participation of both T and B host lymphocytes in the activation of LTβR in these tumors. Tumor growth was also reduced in LIGHT‐deficient mice, suggesting a contribution of this ligand to the activation of LTβR in BFS‐1 fibrosarcomas. LTβR signaling can involve IκBα and/or NFκB‐inducing kinase (NIK) for subsequent NFκB activation in different types of cells. Expression of a dominant negative form of IκBα or of a dominant negative mutant of NIK resulted in decreased activation of NFκB signaling and reduced expression of pro‐angiogenic CXCL2 in vitro. Moreover, expression of dominant negative form of NIK or an IκBα repressor in these fibrosarcoma cells resulted in reduced solid tumor growth in vivo, suggesting that both IκBα and NIK are involved in pro‐angiogenic signaling after LTβR activation. Our data support the idea that the ablation of LTβR signaling should be considered for cancer treatment.     

c‐Jun N‐terminal kinase in pancreatic tumor stroma augments tumor development in mice

Pancreatic ductal adenocarcinoma (PDAC) is a life‐threatening disease and there is an urgent need to develop improved therapeutic approaches. The role of c‐Jun N‐terminal kinase (JNK) in PDAC stroma is not well defined even though dense desmoplastic reactions are characteristic of PDAC histology. We aimed to explore the role of JNK in PDAC stroma in mice. We crossed Ptf1a^Cre/+;Kras^G12D/+ mice with JNK1^?/? mice to generate Ptf1a^Cre/+;Kras^G12D/+;JNK1^?/? (Kras;JNK1^?/?) mice. Tumor weight was significantly lower in Kras;JNK1^?/? mice than in Kras;JNK1^+/? mice, whereas histopathological features were similar. We also transplanted a murine PDAC cell line (mPC) with intact JNK1 s.c. into WT and JNK1^?/? mice. Tumor diameters were significantly smaller in JNK1^?/? mice. Phosphorylated JNK (p‐JNK) was activated in α‐smooth muscle actin (SMA)‐positive cells in tumor stroma, and mPC‐conditioned medium activated p‐JNK in tumor‐associated fibroblasts (TAF) in vitro. Relative expression of Ccl20 was downregulated in stimulated TAF. Ccl20 is an important chemokine that promotes CD8⁺ T‐cell infiltration by recruitment of dendritic cells, and the number of CD8⁺ T cells was decreased in Kras;JNK1^+/? mice compared with Kras;JNK1^?/? mice. These results suggest that the cancer secretome decreases Ccl20 secretion from TAF by activation of JNK, and downregulation of Ccl20 secretion might be correlated with reduction of infiltrating CD8⁺ T cells. Therefore, we concluded that inhibition of activated JNK in pancreatic tumor stroma could be a potential therapeutic target to increase Ccl20 secretion from TAF and induce accumulation of CD8⁺ T cells, which would be expected to enhance antitumor immunity.     

CXCR2‐mediated tumor‐associated neutrophil recruitment is regulated by IFN‐β

The chemokine receptor CXCR2 and its ligands CXCL1, CXCL2 and CXCL5 play an important role in homing of tumor‐associated neutrophils (TANs) into developing tumors. TANs are known to support the development of blood vessels in growing solid tumors, hence contributing to tumor growth. Here, we show that the migration of neutrophils is influenced by endogenous interferon‐beta (IFN‐β) via regulation of such chemokines and their receptor. We could demonstrate that CXCL1 and CXCL2 gradients are formed in tumor‐bearing mice, i.e., low chemokine level in bone marrow (BM) and high level in the tumor. This supports migration of neutrophils into the tumor. Moreover, expression of CXCR2 was highest on neutrophils from BM and lowest in TANs. Importantly, although IFN‐β appears to have only a minor influence on the expression of CXCR2, it strongly regulates the CXCR2 ligands. In the absence of endogenous IFN‐β, they were expressed significantly higher in tumor‐infiltrating neutrophils. Treatment of such neutrophils from tumor‐bearing Ifnb1^?/? mice with recombinant IFN‐β downregulated CXCR2 ligand expression to wild‐type levels. This explains the reduced migration of neutrophils into tumors and the diminished tumor angiogenesis in IFN‐β‐sufficient mice. Our results add a novel functional aspect of the type I IFN system as effector molecules of natural cancer surveillance and open interesting possibilities for antineutrophil therapies against cancer.     

Lack of interleukin‐6 in the tumor microenvironment augments type‐1 immunity and increases the efficacy of cancer immunotherapy

Conquering immunosuppression in tumor microenvironments is crucial for effective cancer immunotherapy. It is well known that interleukin (IL)‐6, a pleiotropic cytokine, is produced in the tumor‐bearing state. In the present study, we investigated the precise effects of IL‐6 on antitumor immunity and the subsequent tumorigenesis in tumor‐bearing hosts. CT26 cells, a murine colon cancer cell line, were intradermally injected into wild‐type and IL‐6‐deficient mice. As a result, we found that tumor growth was decreased significantly in IL‐6‐deficient mice compared with wild‐type mice and the reduction was abrogated by depletion of CD8⁺ T cells. We further evaluated the immune status of tumor microenvironments and confirmed that mature dendritic cells, helper T cells and cytotoxic T cells were highly accumulated in tumor sites under the IL‐6‐deficient condition. In addition, higher numbers of interferon (IFN)‐γ‐producing T cells were present in the tumor tissues of IL‐6‐deficient mice compared with wild‐type mice. Surface expression levels of programmed death‐ligand 1 (PD‐L1) and MHC class I on CT26 cells were enhanced under the IL‐6‐deficient condition in vivo and by IFN‐γ stimulation in vitro. Finally, we confirmed that in vivo injection of an anti‐PD‐L1 antibody or a Toll‐like receptor 3 ligand, polyinosinic‐polycytidylic acid, effectively inhibited tumorigenesis under the IL‐6‐deficient condition. Based on these findings, we speculate that a lack of IL‐6 produced in tumor‐bearing host augments induction of antitumor effector T cells and inhibits tumorigenesis in vivo, suggesting that IL‐6 signaling may be a promising target for the development of effective cancer immunotherapies.