Targeted delivery of oligonucleotides into tumor-associated macrophages for cancer immunotherapy

Tumor-associated macrophages (TAMs) have been proven to be a driving force in the initiation, proliferation, metastasis and angiogenesis of various tumors. Specifically, alterations in the functions of TAMs exhibited inhibitory effects on tumor growth. However, there is currently no research being conducted on the targeting delivery of drugs into TAMs for cell-specific tumor immunotherapy. In the present study, we developed a TAMs targeted delivery system that is triggered by the acidic microenvironment in the tumor to release a TAMs-recognizing nano-complex loaded with oligonucleotides. By using this system, we demonstrated a significant anti-tumor effect of an oligonucleotide combination of CpG oligonucleotide, anti-IL-10 and anti-IL-10 receptor oligonucleotides. These nucleic acid drugs delivered by the delivery system accumulated in the TAMs of an allograft hepatoma murine model by intravenous injection, suppressed the pro-tumor functions and stimulated the anti-tumor activities of TAMs. More importantly, the nucleic acid drug-based immune-regulation was restricted to the tumor microenvironment and did not cause an upregulation of serum inflammatory cytokines. Our present study provides an effective therapeutic strategy for regulating cell-specific functions using nucleic acid drugs.     

Targeting tumor-associated macrophages as a novel strategy against breast cancer

Tumor-associated macrophages (TAMs) are associated with tumor progression and metastasis. Here, we demonstrate for the first time that legumain, a member of the asparaginyl endopeptidase family functioning as a stress protein, overexpressed by TAMs, provides an ideal target molecule. In fact, a legumain-based DNA vaccine served as a tool to prove this point, as it induced a robust CD8+ T cell response against TAMs, which dramatically reduced their density in tumor tissues and resulted in a marked decrease in proangiogenic factors released by TAMs such as TGF-beta, TNF-alpha, MMP-9, and VEGF. This, in turn, led to a suppression of both tumor angiogenesis and tumor growth and metastasis. Importantly, the success of this strategy was demonstrated in murine models of metastatic breast, colon, and non-small cell lung cancers, where 75% of vaccinated mice survived lethal tumor cell challenges and 62% were completely free of metastases. In conclusion, decreasing the number of TAMs in the tumor stroma effectively altered the tumor microenvironment involved in tumor angiogenesis and progression to markedly suppress tumor growth and metastasis. Gaining better insights into the mechanisms required for an effective intervention in tumor growth and metastasis may ultimately lead to new therapeutic targets and better anticancer strategies.     

Bevacizumab and rapamycin inhibit tumor growth in peritoneal model of human ovarian cancer

Ovarian cancer is the leading cause of death from gynecologic cancer. Often, the disease has spread beyond the ovary to involve the peritoneal cavity and causes ascites. Whereas mammalian target of rapamycin (mTOR) functions to regulate protein translation, cell cycle progression, and metastasis, vascular endothelial growth factor promotes tumor angiogenesis, ascites formation, and metastasis in ovarian cancer. In this study, an i.p. model of human ovarian cancer was used to determine the antitumor activity of rapamycin, bevacizumab, and rapamycin plus bevacizumab (BEV/RAPA). We report that administration of rapamycin, bevacizumab, and BEV/RAPA in mice bearing peritoneal OV-90 ovarian carcinoma resulted in 74.6%, 82.4%, and 93.3% reduction in i.p. tumor burden, respectively. BEV/RAPA-induced reduction in microvessel density and inhibition of cell proliferation were associated with significant reduction in hypoxia-inducible factor-1alpha and cyclin D1 and inactivation of downstream targets of mTOR, p70S6 kinase, S6R, and 4E-binding protein 1. BEV/RAPA treatment was not only able to prolong life of i.p. mice but also more effective than rapamycin and bevacizumab to prevent the development of peritoneal carcinomatosis in adjuvant setting and reverse ascites accumulation in heavy peritoneal disease. Our data indicate that simultaneous inhibition of the vascular endothelial growth factor receptor and mTOR pathways with BEV/RAPA or their analogues may represent a novel approach for prevention of metastasis, recurrence, and treatment of ovarian cancer.     

自噬调控巨噬细胞参与构建肿瘤微环境研究进展

自噬作为细胞高度保守的胞内代谢产物及衰老、损伤细胞器的降解途径，参与维持细胞稳态。肿瘤相关巨噬细胞（tumor-associated macrophages，TAMs）是构成肿瘤微环境的关键细胞，通过极化为M2型促进肿瘤发生、发展及转移。自噬通过调控TAMs极化、代谢来参与肿瘤微环境的构建。本文就自噬调控巨噬细胞与肿瘤微环境相互作用的机制进行阐述，旨在为靶向肿瘤微环境内TAMs和肿瘤细胞自噬调控的治疗提供新的理论基础。     

Rare diffuse peritoneal malignant neoplasms: CT findings in two cases

In the peritoneal cavity, diffuse serosal replacement by tumor is demonstrated usually by extensive carcinomatous involvement from gastric, colonic, or pancreatic tumors or less frequently by mesothelioma. Primary tumors other than mesothelioma are extremely rare in the peritoneum. The computed tomographic appearances of two cases of rare peritoneal tumors, epithelioid hemangioendothelioma and desmoplastic small round cell tumor, are described.     

Tumor-associated macrophages in breast cancer as potential biomarkers for new treatments and diagnostics

While several inflammatory cell types participate in cancer development, macrophages specifically play a key role in breast cancer, where they appear to be part of the pathogenesis of high-grade tumors. Tumor-associated macrophages (TAMs) produce factors that promote angiogenesis, remodel tissue and dampen the immune response to tumors. Specific macrophage types contribute to increased metastases in animal models, while human studies show an association between TAMs and tumors with poor prognostic features. Macrophages display a spectrum of phenotypic states, with the tumor microenvironment skewing TAMs towards a 'nonclassical' activation state, known as the M2, or wound healing/regulatory state. These TAMs are found in high-risk breast cancers, making them an important therapeutic target to explore. Improved techniques for identifying TAMs should translate into clinical applications for prognosis and treatment.     

肿瘤相关巨噬细胞与肿瘤耐药及治疗研究进展

肿瘤耐药的产生是肿瘤细胞与肿瘤微环境(tumor microenvironment, TME)相互作用的结果, 肿瘤相关巨噬细胞(tumor-associated macrophages, TAMs)是TME中的主要免疫细胞, 在炎症微环境和肿瘤细胞的恶性表型之间发挥桥梁作用, 与肿瘤耐药和疾病进展密切相关, 其中M2型TAMs浸润则预示着不良的临床结局。本文主要针对TAMs参与肿瘤耐药的作用机制和治疗进展进行综述, 以期为减少肿瘤耐药、增强抗肿瘤治疗疗效提供参考。     

Intratumoral poly-N-acetyl glucosamine-based polymer matrix provokes a prolonged local inflammatory response that, when combined with IL-2, induces regression of malignant mesothelioma in a murine model

The authors have previously shown that cytokines delivered directly into malignant mesothelioma (MM) tumors can retard tumor growth and mediate tumor regression under certain conditions. In this report the authors compared the efficacy of serial intratumoral injections of three cytokines, GM-CSF, IL-12, and IL-2, to their sustained release using a single injection in a poly-N-acetyl glucosamine gel. IL-2 combined with the polymer gel gave optimal antitumor results when MM tumors were accessible as either subcutaneous deposits or as masses spread throughout the peritoneal cavity. The gel acted not only as a slow-release cytokine depot but also as a trigger for inflammation and recruited several immune cell types to the gel/tumor interface; when combined with IL-2 (but not with GM-CSF or IL-12), it acted as a selective reservoir for infiltrating CD8+ T cells. Hence, the IL-2/gel may provide a microenvironment that allows intratumoral T cells to proliferate and retain their cytolytic functions as they encounter their cognate antigens expressed by tumor cells.     

腹膜恶性间皮瘤的超声表现及其诊断价值

目的探讨腹膜恶性间皮瘤的超声表现及超声诊断价值。方法回顾性分析20例腹膜恶性间皮瘤的超声表现，并与8例腹膜转移癌及5例结核性腹膜炎声像图对比分析。结果腹膜间皮瘤超声表现为腹膜弥漫或局限性增厚，腹膜上可见低回声小结节，部分患者腹水内见大量强回声分隔。腹膜转移癌及结核性腹膜炎声像图与其相似。结论恶性间皮瘤的超声表现不具特异性，很难与腹膜转移癌及结核性腹膜炎相互鉴别，超声引导下对腹膜增厚区或腹膜可疑结节穿刺活检及腹腔镜腹膜活检是安全有效的诊断方法。     

RANK ligand inhibition plus docetaxel improves survival and reduces tumor burden in a murine model of prostate cancer bone metastasis

Tumor cells induce excessive osteoclastogenesis, mediating pathologic bone resorption and subsequent release of growth factors and calcium from bone matrix, resulting in a "vicious cycle" of bone breakdown and tumor proliferation. RANK ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival. In metastatic prostate cancer models, RANKL inhibition directly prevents osteolysis via blockade of osteoclastogenesis and indirectly reduces progression of skeletal tumor burden by reducing local growth factor and calcium concentrations. Docetaxel, a well-established chemotherapy for metastatic hormone-refractory prostate cancer, arrests the cell cycle and induces apoptosis of tumor cells. Suppression of osteoclastogenesis through RANKL inhibition may enhance the effects of docetaxel on skeletal tumors. We evaluated the combination of the RANKL inhibitor osteoprotegerin-Fc (OPG-Fc) with docetaxel in a murine model of prostate cancer bone metastasis. Tumor progression, tumor area, and tumor proliferation and apoptosis were assessed. OPG-Fc alone reduced bone resorption (P < 0.001 versus PBS), inhibited progression of established osteolytic lesions, and reduced tumor area (P < 0.0001 versus PBS). Docetaxel alone reduced tumor burden (P < 0.0001 versus PBS) and delayed the development of osteolytic lesions. OPG-Fc in combination with docetaxel suppressed skeletal tumor burden (P = 0.0005) and increased median survival time by 16.7% (P = 0.0385) compared with docetaxel alone. RANKL inhibition may enhance docetaxel effects by increasing tumor cell apoptosis as evident by increased active caspase-3. These studies show that inhibition of RANKL provides an additive benefit to docetaxel treatment in a murine model of prostate cancer bone metastasis and supports clinical evaluation of this treatment option in patients.     

LY2109761, a novel transforming growth factor beta receptor type I and type II dual inhibitor, as a therapeutic approach to suppressing pancreatic cancer metastasis

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor beta (TGF-beta) plays a key role in cancer metastasis, signaling through the TGF-beta type I/II receptors (TbetaRI/II). We hypothesized that targeting TbetaRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-beta1-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TbetaRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TbetaRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis.     

Neutralizing monoclonal antibody to periostin inhibits ovarian tumor growth and metastasis

Periostin, an extracellular matrix protein, is reported to be overexpressed in a variety of human cancers and its functions seem to be linked to tumor metastasis. Our previous results show that engineered periostin overexpression promotes ovarian tumor growth and dissemination in vivo. In this study, we developed a neutralizing monoclonal antibody to periostin, named MZ-1, and investigated its effects on human ovarian tumor growth and metastasis. Our in vivo studies showed significant growth inhibition by MZ-1 on both subcutaneous and intraperitoneal (i.p.) tumors derived from the periostin-expressing ovarian cancer cell line A2780. In addition, MZ-1 treatment led to a reduction of the metastatic potential of these A2780 i.p. tumors. The in vivo antitumor effects of MZ-1 were linked to its specific inhibition of anchorage-independent growth and survival of periostin-expressing cells, as well as its neutralizing effects on periostin-induced cancer cell migration and invasion. The data suggest that blocking periostin expression may be a novel approach for treating the subset of invasive ovarian tumors that overexpress periostin protein.     

"Re-educating" tumor-associated macrophages by targeting NF-kappaB

The nuclear factor kappaB (NF-kappaB) signaling pathway is important in cancer-related inflammation and malignant progression. Here, we describe a new role for NF-kappaB in cancer in maintaining the immunosuppressive phenotype of tumor-associated macrophages (TAMs). We show that macrophages are polarized via interleukin (IL)-1R and MyD88 to an immunosuppressive "alternative" phenotype that requires IkappaB kinase beta-mediated NF-kappaB activation. When NF-kappaB signaling is inhibited specifically in TAMs, they become cytotoxic to tumor cells and switch to a "classically" activated phenotype; IL-12(high), major histocompatibility complex II(high), but IL-10(low) and arginase-1(low). Targeting NF-kappaB signaling in TAMs also promotes regression of advanced tumors in vivo by induction of macrophage tumoricidal activity and activation of antitumor activity through IL-12-dependent NK cell recruitment. We provide a rationale for manipulating the phenotype of the abundant macrophage population already located within the tumor microenvironment; the potential to "re-educate" the tumor-promoting macrophage population may prove an effective and novel therapeutic approach for cancer that complements existing therapies.     

Intracellular uptake and intracavitary targeting of folate-conjugated liposomes in a mouse lymphoma model with up-regulated folate receptors

The folate receptor is overexpressed in a broad spectrum of malignant tumors and represents an attractive target for selective delivery of anticancer agents to folate receptor-expressing tumors. This study examines folate-lipid conjugates as a means of enhancing the tumor selectivity of liposome-encapsulated drugs in a mouse lymphoma model. Folate-derivatized polyethylene glycol (PEG3350)-distearoyl-phosphatidylethanolamine was post-loaded at various concentrations into the following preparations: radiolabeled PEGylated liposomes, PEGylated liposomes labeled in the aqueous compartment with dextran fluorescein, and PEGylated liposomal doxorubicin (PLD, Doxil). We incubated folate-targeted radiolabeled or fluorescent liposomes with mouse J6456 lymphoma cells up-regulated for their folate receptors (J6456-FR) to determine the optimal ligand concentration required in the lipid bilayer for liposomal cell association, and to examine whether folate-targeted liposomes are internalized by J6456-FR cells in suspension. Liposomal association with cells was quantified based on radioactivity and fluorescence-activated cell sorting analysis, and internalization was assessed by confocal fluorescence microscopy. We found an optimal ligand molar concentration of approximately 0.5% using our ligand. A substantial lipid dose-dependent increase in cell-associated fluorescence was found in folate-targeted liposomes compared with nontargeted liposomes. Confocal depth scanning showed that a substantial amount of the folate-targeted liposomes are internalized by J6456-FR cells. Binding and uptake of folate-targeted PLD by J6456-FR cells were also observed in vivo after i.p. injection of folate-targeted PLD in mice bearing ascitic J6456-FR tumors. The drug levels in ascitic tumor cells were increased by 17-fold, whereas those in plasma were decreased by 14-fold when folate-targeted PLD were compared with nontargeted PLD in the i.p. model. Folate-targeted liposomes represent an attractive approach for the intracellular delivery of drugs to folate receptor-expressing lymphoma cells and seem to be a promising tool for in vivo intracavitary drug targeting.     

Efficacy of S-1 for patients with peritoneal metastasis of gastric cancer

BACKGROUND: This study was designed to examine the efficacy and compliance of S-1 for the patients with peritoneal metastasis of gastric cancer. METHODS: Sixteen consecutive patients with peritoneal metastasis of gastric cancer were treated with S-1. Their survival was compared with that of the historical control group (25 patients). Thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase and orotate phosphoribosyl transferase mRNA expression in the tumor were evaluated. RESULTS: The median survival time of S-1-treated patients was 550 days, which was significantly longer than that of the historical control group (215 days). We elucidated some factors to prolong the survival of the patients treated with S-1 for peritoneal metastasis: peritoneal metastasis without other distant metastases, the combination of S-1 treatment and gastrectomy, and low expression of thymidine phosphorylase mRNA in primary tumors. Conclusions: S-1 showed a surprisingly long-term survival with minimum toxicity in patients with peritoneal metastasis of gastric cancer.     

IL-15 protects NKT cells from inhibition by tumor-associated macrophages and enhances antimetastatic activity

Vα24-invariant NKT cells inhibit tumor growth by targeting tumor-associated macrophages (TAMs). Tumor progression therefore requires that TAMs evade NKT cell activity through yet-unknown mechanisms. Here we report that a subset of cells in neuroblastoma (NB) cell lines and primary tumors expresses membrane-bound TNF-α (mbTNF-α). These proinflammatory tumor cells induced production of the chemokine CCL20 from TAMs via activation of the NF-κB signaling pathway, an effect that was amplified in hypoxia. Flow cytometry analyses of human primary NB tumors revealed selective accumulation of CCL20 in TAMs. Neutralization of the chemokine inhibited in vitro migration of NKT cells toward tumor-conditioned hypoxic monocytes and localization of NKT cells to NB grafts in mice. We also found that hypoxia impaired NKT cell viability and function. Thus, CCL20-producing TAMs served as a hypoxic trap for tumor-infiltrating NKT cells. IL-15 protected antigen-activated NKT cells from hypoxia, and transgenic expression of IL-15 in adoptively transferred NKT cells dramatically enhanced their antimetastatic activity in mice. Thus, tumor-induced chemokine production in hypoxic TAMs and consequent chemoattraction and inhibition of NKT cells represents a mechanism of immune escape that can be reversed by adoptive immunotherapy with IL-15-transduced NKT cells.     

Oncolytic Adenovirus Expressing Soluble TGF�� Receptor II-Fc-mediated Inhibition of Established Bone Metastases: A Safe and Effective Systemic Therapeutic Approach for Breast Cancer

In recent years, oncolytic adenoviruses have shown some promise as a novel class of antitumor agents. However, their utility in targeting bone metastases is relatively less studied. We have examined whether the systemic therapy of oncolytic adenoviruses expressing the soluble form of transforming growth factor-β (TGFβ) receptor II fused with human immunoglobulin G1 can be developed for the treatment of established breast cancer bone metastases. MDA-MB-231-luc2 human breast cancer cells were injected in the left heart ventricle of nude mice to establish bone metastasis. Mice with hind limb tumors were administered (on days 8 and 11) oncolytic adenoviruses-Ad.sTβRFc or mhTERTAd.sTβRFc. Skeletal tumor growth was monitored weekly by bioluminescence imaging (BLI) and radiography. At the termination time on day 28, hind limb bones were analyzed for tumor burden, synchrotron micro-computed tomography, and osteoclast activation. Intravenous delivery of Ad.sTβRFc and mhTERTAd.sTβRFc induced significant inhibition of tumor growth, reduction of tumor burden, osteoclast activation, and increased animals'' survival. Oncolytic adenoviruses were safer than dl309, a wild-type virus. A slight elevation of liver enzyme activity was observed after Ad.sTβRFc administration; this subsided with time. Based on these studies, we believe that Ad.sTβRFc and mhTERTAd.sTβRFc can be developed as a safe and effective approach for the treatment of established bone metastasis.     

Countering tumor-induced immunosuppression during immunotherapy for pancreatic cancer

Background: Vaccines for pancreatic cancer have been challenged by a number of factors, especially the immunosuppressive microenvironment within the tumor that allows for escape from immune surveillance. Objective/methods: We sought to identify results that define mechanisms of pancreatic-cancer-associated immunosuppression and strategies that might be useful to overcome them thereby resulting in effective immune responses to cancer vaccines capable of deleting pancreatic cancer cells. Results/conclusion: Immunosuppressive tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg) reside in tumors, and their products along with tumor derived products (such as VEGF, TGFβ and IL-10), create a microenvironment that counters immune activation and attack. Immunotherapy with cancer vaccines must include strategies to modulate these immunosuppressive cell types and tumor byproducts. Clinical trials are beginning to test these strategies.     

An acid trip activates protumoral macrophages to promote hepatocellular carcinoma malignancy

Tumor-associated macrophages (TAMs) promote metastasis and tumor cell extravasation, survival, and growth. In hepatocellular carcinoma (HCC), the presence of TAM subpopulations correlates with poor outcome. In this issue of the JCI, Ning et al. report on their use of cell culture, mouse models, and human data sets to investigate the interactions between aerobic glycolysis and carbonic anhydrase XII (CA12) expression in HCC. Aerobic glycolysis promoted CA12 upregulation in TAMs, which induced a protumoral phenotype to promote tumor growth and metastasis. Tumor cell factors derived from HCC samples induced CA12 upregulation in tumor-infiltrating TAMs via the HIF1α pathway. In preclinical models of HCC, CA12 inhibition reduced tumor growth and lung metastasis and reduced TAM infiltrate. Notably, dual treatment with anti-PD1 and CA12 inhibitors synergistically attenuated tumor growth and metastasis and enhanced survival compared with either treatment alone. These findings suggest that targeting CA12 in combination with immune-checkpoint blockade may provide treatment options for HCC.