Generation of a Dual-Functioning Antitumor Immune Response in the Peritoneal Cavity

Tumor cell metastasis to the peritoneal cavity is observed in patients with tumors of peritoneal organs, particularly colon and ovarian tumors. Following release into the peritoneal cavity, tumor cells rapidly attach to the omentum, a tissue consisting of immune aggregates embedded in adipose tissue. Despite their proximity to potential immune effector cells, tumor cells grow aggressively on these immune aggregates. We hypothesized that activation of the immune aggregates would generate a productive antitumor immune response in the peritoneal cavity. We immunized mice i.p. with lethally irradiated cells of the colon adenocarcinoma line Colon38. Immunization resulted in temporary enlargement of immune aggregates, and after challenge with viable Colon38 cells, we did not detect tumor growth on the omentum. When Colon38-immunized mice were challenged with cells from the unrelated breast adenocarcinoma line E0771 or the melanoma line B16, these tumors also did not grow. The nonspecific response was long-lived and not present systemically, highlighting the uniqueness of the peritoneal cavity. Cellular depletions of immune subsets revealed that NK1.1⁺ cells were essential in preventing growth of unrelated tumors, whereas NK1.1⁺ cells and T cells were essential in preventing Colon38 tumor growth. Collectively, these data demonstrate that the peritoneal cavity has a unique environment capable of eliciting potent specific and nonspecific antitumor immune responses.The peritoneal cavity is a unique immunologic environment that includes immune aggregates present in the peritoneal wall, mesentery, and omentum as well as free cells present in the peritoneal fluid.^1,2 This fluid, which mechanically acts to lubricate organ movement, also distributes a variety of immune subsets throughout the peritoneal cavity. The immune cells present in the peritoneal fluid are primarily macrophages and B cells but also include other lymphocyte and dendritic cell populations.³ These free-floating immune cells have a dynamic relationship with the organized immune aggregates also present in the peritoneal cavity.^4,5 These structures contain immune cell subsets similar to those in the peritoneal fluid but in a highly organized manner, similar to many other tertiary immune structures.^3,6,7 One site of these immune aggregates, the omentum, is of particular interest because of the high density of aggregates found there.The omentum is a thin adipose tissue located in the peritoneal cavity that is appreciated as a guardian of the peritoneal cavity, especially for its immunologic role in controlling infections. For example, peritoneal dialysis, which can introduce bacteria into the cavity, leads to increases in the number and size of omental immune aggregates, which further increase on complications of peritonitis.^8,9 In addition, omental immune aggregates are the primary site of leukocyte extravasation in models of peritonitis.^10,11 Furthermore, bacteria are rapidly sequestered in the omentum shortly after introduction to the peritoneal cavity,¹² a process that slows bacterial dissemination throughout the peritoneal cavity.⁸ Collectively, these data suggest that omental immune aggregates are capable of responding against foreign pathogens.Similar to bacterial localization to the omentum, following tumor cell metastasis to the peritoneal cavity, the initial and most common site of tumor formation is the omentum.⁷ Tumor cell metastasis to the peritoneal cavity is generally a poor prognostic indicator, and limited effective therapies are available to diagnosed individuals.^13,14 Omental metastasis is a common occurrence in individuals diagnosed as having cancers of the ovary and colon as well as other peritoneal organs.^15,16 It is specifically immune aggregates to which metastasizing cells originally bind and subsequently divide.⁷ Tumor growth on the omentum is suggested to be a result of preferential binding to this site and the presence of factors that promote tumor growth.^7,17,18 After tumor formation on the omentum, tumor cells often further disseminate to other sites in the peritoneal cavity, as well as systemically, further propagating disease.Despite data demonstrating the immune capabilities of the omentum,^4,6 the omental immune response to tumor metastasis is relatively understudied. Limited work shows that after cells adhere to the omentum, the vasculature of omental immune aggregates is well-suited to supporting rapid tumor growth. Under normal conditions, the vasculature of omental immune aggregates exhibits a phenotype that may be capable of rapid expansion after an immunologic stimulus, which is exploited by metastasizing tumor cells.⁷ Despite the abundance of immune cells present at the site of tumor growth, a productive immune response does not occur naturally, and tumors grow progressively.^3,19In an attempt to determine whether the omental immune microenvironment is capable of promoting antitumor responses, we immunized mice with lethally irradiated tumor cells. Because the omentum is the initial site of tumor cell binding, i.p. immunization with these lethally irradiated tumor cells allows us to target the omentum to potentially generate an antitumor immune response. Herein, we found that i.p. immunization with lethally irradiated tumor cells led to the production of an antitumor immune response that was effective in controlling the growth of both specific and unrelated tumors after a secondary challenge with viable tumor cells. The nonspecific antitumor response was unique to the peritoneal cavity and was sustained for ≥60 days after immunization. In addition, depletion of NK1.1⁺ cells reversed the protective effects elicited by immunization only when challenged with an unrelated tumor challenge. In contrast, depletion of NK1.1⁺ cells and conventional T-cell populations was required to reverse the protective effects against specific tumor challenge. Thus, activation of peritoneal NK1.1⁺ cells in addition to conventional T-cell populations may have potent antitumor capabilities that could be exploited to benefit patients therapeutically.     

Preferential attachment of peritoneal tumor metastases to omental immune aggregates and possible role of a unique vascular microenvironment in metastatic survival and growth

Controlling metastases remains a critical problem in cancer biology. Within the peritoneal cavity, omental tissue is a common site for metastatic disease arising from intraperitoneal tumors; however, it is unknown why this tissue is so favorable for metastatic tumor growth. Using five different tumor cell lines in three different strains of mice, we found that the omentum was a major site of metastases growth for intraperitoneal tumors. Furthermore, initial attachment and subsequent growth were limited to specific sites within the omentum, consisting of organized aggregates of immune cells. These immune aggregates contained a complex network of capillaries exhibiting a high vascular density, which appear to contribute to the survival of metastatic cells. We found that the vasculature within these aggregates contained CD105+ vessels and vascular sprouts, both indicators of active angiogenesis. A subset of mesothelial cells situated atop the immune aggregates was found to be hypoxic, and a similar proportion was observed to secrete vascular endothelial growth factor-A. These data provide a physiological mechanism by which metastatic tumor cells preferentially grow at sites rich in proangiogenic vessels, apparently stimulated by angiogenic factors produced by mesothelial cells. These sites provide metastatic cells with a microenvironment highly conducive to survival and subsequent growth.     

Comparative light and electron microscopic studies of cystic and papillary tumors of the peritoneum

Summary Using a cystic lymphangioma of the greater omentum and a benign cystic mesothelioma as examples, the distinguishing characteristics of cystic peritoneal tumors are shown, using light microscopic and ultrastructural findings. A benign papillary mesothelioma is used for comparison. The cellular structures and growth rate of the mesotheliomas are contrasted with the tissue reactions which are typical for irritated serosa. The diffusely growing papillary mesothelioma is a very rare tumor, the cells of which are similar to normal serosa cells, but also show characteristics of other benign or malignant mesothelial tumors and of reactive proliferated mesothelial cells. The diffusely growing benign cystic mesothelioma has only been described in isolated cases and is characterized by cysts grouped in acini with mainly flat, localized cuboidal cell lining. The histochemical properties and cytological findings correspond closely to those of the papillary tumor or normal peritoneal lining cells. In contrast, the cystic lymphangioma probably represents a congenital defect with a slow growth rate. The structure is characterized by a sponge-like arrangement of smooth-walled cysts, in the walls of which smooth muscle cells and lymph follicles are embedded. The endothelium is also flat and ultrastructurally resembles that of lymph vessels.     

Interleukin-6 receptor enhances early colonization of the murine omentum by upregulation of a mannose family receptor,LY75, in ovarian tumor cells

One of the earliest metastatic events in human ovarian cancer, tumor spread to the omentum, may be influenced by expression of interleukin 6 (IL6) and its cognate receptor (IL6Rα). Previous reports have shown that IL6 and IL6Rα expression is elevated in the serum and ascites of patients with ovarian cancer and that this can influence in vitro processes such as cell survival, proliferation and migration. In this study, overexpression of IL6Rα, and to a lesser extent IL6, enhanced tumor growth on the omentum. Moreover, adherence to plastic and to peritoneal extracellular matrix components was enhanced in tumor cells overexpressing IL6 or IL6Rα. Host production of IL6 and IL6Rα was also sufficient to influence tumor adherence to the omentum. Expression of LY75/CD205/DEC205, a collagen-binding mannose family receptor, was directly influenced by IL6Rα expression. Blocking LY75 with antibody reduced the adherence of tumor cells overexpressing IL6Rα to matrices in vitro and to the omentum. The association between IL6Rα expression and LY75 expression has not been previously reported, and the promotion of cellular adherence is a novel role for LY75. These studies indicate that overexpression of LY75 may be an additional mechanism by which IL6 signaling influences the progression of ovarian cancer, and suggests that blocking LY75 could be a valuable clinical strategy for reducing the early metastasis of ovarian cancer.     

Complement-dependent inhibition of degradation of soluble immune complexes and immunoglobulin aggregates by thioglycollate-stimulated peritoneal macrophages.

Previous studies have shown that the degradation of soluble immune complexes or aggregates (AIgG) by normal peritoneal macrophages can be enhanced by complement. The enhancement of degradation was shown to be at least in part dependent on the number of C3b molecules bound per complex. The present investigations indicate that the enhanced degradation is not found with thioglycollate-stimulated macrophages, and that at high concentrations of complement, inhibition may even occur. The Fc receptor-mediated degradation of soluble immune complexes and AIgG by stimulated macrophages was at least twice as high as that by normal macrophages. This increase was compatible with the increased number of Fc receptors on the stimulated macrophages. The inhibitory effect of high concentrations of serum, as a complement source, on the degradation of AIgG was dependent on the number of C3b molecules bound per AIgG. Although there was also a two-fold increase in the number of C3b receptor sites on the stimulated macrophages, more than 11 C3b molecules per AIgG40 caused significant inhibition of degradation. This phenomenon may be dependent on shielding of Fc-Fc receptor interaction by varying numbers of C3b molecules per complex.     

卵巢肿瘤继发腹膜黑色素沉着症的临床病理观察

目的探讨卵巢肿瘤继发腹膜黑色素沉着症的临床病理学特点和生物学行为。方法对3例卵巢肿瘤继发腹膜黑色素沉着症作了临床病理观察、免疫组化染色,并作了随访。结果3例患者年龄分别为17、29和23岁。例1卵巢囊性肿物,腹腔内多个大小不等的灰褐色肿物,切面囊内充满墨汁样液体。例2做了卵巢肿瘤姑息手术,术中检查腹腔和盆腔内有浓稠的墨汁样黑色素沉着。例3瘤体内有皮脂、毛发和充盈黑色液体的囊性结节。镜检:除例1伴发浆液性囊腺瘤,例3伴发囊性畸胎瘤外,3例病变基本相似,主要由胞质充满黑色素的细胞和胞质内无色素的细胞组成。绝大部分区域以前者占优势,纤维组织增生并纤维化,大量色素沉着使原有结构被掩盖。例1见少量破骨样多核巨细胞,腹膜和网膜内亦有大量黑色素沉着和纤维组织增生。例3Fontana染色阳性,普鲁士蓝染色阴性。例1免疫组化染色胞质内含有色素颗粒和无色素颗粒的细胞均有部分细胞HMB-45阳性,浆液性囊腺瘤内衬的上皮CK呈阳性,胞质内含有色素颗粒和无色素颗粒的细胞CK呈阴性,S-100、CD68、MC、CD34、ER、PR和NSE均阴性。结论腹膜黑色素沉着症为卵巢肿瘤和胃肠道发育畸形继发的一种良性病变。     

Inhibition of adhesion and proliferation of peritoneally disseminated tumor cells by pegylated catalase

Hydrogen peroxide may aggravate the peritoneal dissemination of tumor cells by activating the expression of a variety of genes. In this study, we used pegylated catalase (PEG-catalase) to examine whether prolonged retention of catalase activity within the peritoneal cavity is effective in inhibiting peritoneal dissemination in mouse models. Murine B16-BL6 cells or colon 26 cells labeled with firefly luciferase gene were inoculated intraperitoneally into syngeneic mice. Compared with unmodified catalase, PEG-catalase was retained in the peritoneal cavity for a long period after intraperitoneal injection. A single injection of PEG-catalase just before tumor inoculation significantly reduced the number of the tumor cells at 1 and 7 days. The changes in the expression of molecules involved in the metastasis were evaluated by real time quantitative PCR analysis. Inoculation of the tumor cells increased the expression of intercellular adhesion molecule (ICAM)-1 in the greater omentum, which was inhibited by PEG-catalase. An injection of PEG-catalase at 3 days after tumor inoculation also reduced the number of the tumor cells, suggesting that processes other than the adhesion of tumor cells to peritoneal organs are also inhibited. Daily doses of PEG-catalase significantly prolonged the survival time of tumor-bearing mice. These results indicate that intraperitoneal injection of PEG-catalase inhibits the multiple processes of peritoneal dissemination of tumor cells by scavenging hydrogen peroxide in the peritoneal cavity.     

The metastasis-promoting roles of tumor-associated immune cells

Tumor metastasis is driven not only by the accumulation of intrinsic alterations in malignant cells, but also by the interactions of cancer cells with various stromal cell components of the tumor microenvironment. In particular, inflammation and infiltration of the tumor tissue by host immune cells, such as tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells, have been shown to support tumor growth in addition to invasion and metastasis. Each step of tumor development, from initiation through metastatic spread, is promoted by communication between tumor and immune cells via the secretion of cytokines, growth factors, and proteases that remodel the tumor microenvironment. Invasion and metastasis require neovascularization, breakdown of the basement membrane, and remodeling of the extracellular matrix for tumor cell invasion and extravasation into the blood and lymphatic vessels. The subsequent dissemination of tumor cells to distant organ sites necessitates a treacherous journey through the vasculature, which is fostered by close association with platelets and macrophages. Additionally, the establishment of the pre-metastatic niche and specific metastasis organ tropism is fostered by neutrophils and bone marrow-derived hematopoietic immune progenitor cells and other inflammatory cytokines derived from tumor and immune cells, which alter the local environment of the tissue to promote adhesion of circulating tumor cells. This review focuses on the interactions between tumor cells and immune cells recruited to the tumor microenvironment and examines the factors allowing these cells to promote each stage of metastasis.     

Sympathetic nerve tissue in milky spots of the human greater omentum

Omental milky spots (OMSs), small lymphoid structures positioned in the greater omentum, are involved in peritoneal immune homeostasis and the formation of omental metastases. Sympathetic nerve activity is known to regulate immune function in other lymphoid organs (e.g. spleen and lymph nodes) and to create a favourable microenvironment for various tumour types. However, it is still unknown whether OMSs receive sympathetic innervation. Therefore, the aim of this study was to establish whether OMSs of the adult human greater omentum receive sympathetic innervation. A total of 18 OMSs were isolated from five omenta, which were removed from 3% formaldehyde-perfused cadavers (with a median age of 84 years, ranging from 64 to 94). OMSs were embedded in paraffin, cut and stained with a general (PGP9.5) and sympathetic nerve marker (TH and DBH), and evaluated by bright field microscopy. A T-cell, B-cell, and macrophage staining was performed to confirm OMS identity. In 50% of the studied OMSs, sympathetic nerve fibres were observed at multiple levels of the same OMS. Nerve fibres were represented as dots or elongated structures and often observed in relation to small vessels and occasionally as individual structures residing between lymphoid cells. The current study shows that 50% of the investigated OMSs contain sympathetic nerve fibres. These findings may contribute to our understanding of neural regulation of peritoneal immune response and the involvement of OMSs in omental metastases.     

Enhanced degradation of soluble immunoglobulin aggregates by macrophages in the presence of complement

The role of complement in the processing of soluble immune complexes by guinea-pig peritoneal macrophages was studied in a homologous system in vitro, using isolated stable IgG2 aggregates as a model for immune complexes. Under serum-free conditions, peritoneal macrophages were already able to degrade substantial amounts of the available immunoglobulin aggregates. Addition of fresh serum to the incubation mixtures caused a marked increase in the rate of degradation. The stimulating effect of fresh serum was complement-mediated, because it was abolished by heat treatment or CoVF treatment of the serum and was not seen when C4 or C3-deficient sera were tested. Reconstitution of C4 and C3-deficient sera with purified C4 or C3 restored the stimulating effect of serum on the degradation of the aggregates. The results suggest that activation of the complement system by immune aggregates enhances binding of the aggregates to the cells, which results in the increased degradation observed.     

Milky Spots Promote Ovarian Cancer Metastatic Colonization of Peritoneal Adipose in Experimental Models

The goal of controlling ovarian cancer metastasis formation has elicited considerable interest in identifying the tissue microenvironments involved in cancer cell colonization of the omentum. Omental adipose is a site of prodigious metastasis in both ovarian cancer models and clinical disease. This tissue is unusual for its milky spots, comprised of immune cells, stromal cells, and structural elements surrounding glomerulus-like capillary beds. The present study shows the novel finding that milky spots and adipocytes play distinct and complementary roles in omental metastatic colonization. In vivo assays showed that ID8, CaOV3, HeyA8, and SKOV3ip.1 cancer cells preferentially lodge and grow within omental and splenoportal fat, which contain milky spots, rather than in peritoneal fat depots. Similarly, medium conditioned by milky spot–containing adipose tissue caused 75% more cell migration than did medium conditioned by milky spot–deficient adipose. Studies with immunodeficient mice showed that the mouse genetic background does not alter omental milky spot number and size, nor does it affect ovarian cancer colonization. Finally, consistent with the role of lipids as an energy source for cancer cell growth, in vivo time-course studies revealed an inverse relationship between metastatic burden and omental adipocyte content. Our findings support a two-step model in which both milky spots and adipose have specific roles in colonization of the omentum by ovarian cancer cells.CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.It is estimated that 22,240 women will be diagnosed with and 14,030 women will die of cancer of the ovary in 2013 (http://seer.cancer.gov/csr/1975_2009_pops09, last accessed June 18, 2013). The majority of patients present with metastases or eventually die of metastatic disease within the abdominal cavity. After escape from the primary tumor, ovarian cancer cells in the peritoneal fluid have access to and can potentially lodge within a variety of tissues.^1,2 In both clinical disease and experimental models, however, the omentum is the site of prodigious metastasis formation.^3–6 Thus, attachment of ovarian cancer cells to the omentum represents an early step in the development of widespread peritoneal disease.^7,8 Although the importance of the omentum is widely acknowledged, there still is no consensus on its role in metastasis formation. This raises the question of what components of the omental tissue microenvironment participate in, or facilitate, ovarian cancer metastatic colonization.Studies of omental function date back to the early 19th century. Jobert de Lamballe, a 19th-century surgeon in France, was reportedly the first to recognize the curious ability of this organ to fight infection and form adhesions to help control injuries.⁹ After nearly two centuries of investigation, a great deal is known about the physiology and surgical applications of the omentum.^9–12 As the central regulator of peritoneal homeostasis, its functions include regulating fluid and solute transport, sensing and repairing injuries, promoting angiogenesis, fighting infection, serving as a source of stem cells, producing regulatory molecules, and storing and supplying lipids. These diverse functions are conferred by the cellular composition and architecture characteristic of human omenta.Aside from the clear collagenous membrane that acts as a scaffold for the organ, the majority of the omentum is composed of bands of adipose tissue that contain adipocytes, blood and lymph vessels, immune cells, stromal cells, and connective matrix components that lie beneath an irregular mesothelium.¹³ In general, adipocytes have a variety of functions, ranging from lipid storage to production of endocrine molecules, and can serve as an integrating hub for inflammation, metabolism, and immunity.^2,14–23 A distinctive feature of the omental vasculature is the presence of numerous branching blood vessels ending in tortuous glomerulus-like capillary beds near the tissue periphery.^24–28 Immune cells aggregate around and within these capillary beds to form milky spots, which are the major immune structure for host defense of the peritoneal cavity.^24,29–36 In milky spots, both the endothelial lining of the capillaries and the overlying mesothelium are specially adapted to facilitate transmigration of immune cells.^24,37,38 Additional structural elements include plasmocytes, fibroblasts, and mesenchymal cells, as well as collagen and reticular and elastic fibers.^29,34,37,39A comprehensive literature review showed that studies examining the role of the omentum in metastasis focus on the contribution of its individual components, and not on the tissue as a whole. In our view, results from the majority of studies support models in which ovarian cancer metastatic colonization is driven either purely by milky spots or purely by adipocytes. The milky spot–driven model is based on a large body of in vivo data showing that, on intraperitoneal injection, cancer cells rapidly and specifically localize, invade, and proliferate within omental milky spots.^{3,6,24,28,40–44} In contrast, the adipocyte-driven model is based on the observation that, in its resting state, the omentum is composed predominantly of adipose and that cultured adipocytes can produce adipokines capable of promoting ovarian cancer cell migration and invasion in vitro.⁴⁵ Adipocytes can also provide a proliferative advantage by transferring fatty acids to ovarian cancer cells.⁴⁵ Although both models have clear strengths, neither addresses the intimate and dynamic interaction among milky spots, surrounding adipocytes, and other components of omental tissues.Taking tissue architecture and function as a guide, we propose that an alternative, more fully integrated model of metastatic colonization is needed. To test this idea, we identified peritoneal fat depots (omentum, mesentery, and uterine, gonadal, and splenoportal fat) that are accessible to ovarian cancer cells after intraperitoneal injection.² Of these, the omentum and splenoportal fat are reported to contain milky spot structures.^24,46 We reasoned that a comparison of peritoneal adipose that either contains or lacks milky spots could be used to determine the contributions of adipocytes and milky spots to the lodging and progressive growth of ovarian cancer cells in physiologically relevant tissues. In vivo studies using a panel of ovarian cancer cell lines showed that milky spots dramatically enhance early cancer cell lodging on peritoneal adipose tissues. Consistent with this finding, conditioned medium from milky spot–containing adipose tissue had a significantly increased ability to direct cell migration, compared with conditioned medium from milky spot–deficient adipose tissue. Studies using a panel of immunodeficient mice showed that the number and size of omental milky spots is not dependent on the mouse genetic background and, similarly, that ovarian cancer cell colonization does not depend on the immune composition of the milky spot. Finally, consistent with the role for lipids as an energy source for ovarian cancer cell growth, in vivo time-course studies revealed an inverse relationship between metastatic burden and adipocyte content in the omentum. Our findings support a two-step model in which both milky spots and adipose have specific roles in colonization of the omentum by ovarian cancer cells.     

Aging decreases beta-endorphin enhancement of T-cell mitogenesis in mice

Numerous studies have demonstrated the role of the central nervous system in immunomodulation. beta-Endorphin, a neuropeptide that is released along with adrenocorticotropin by the pituitary in response to stress, has been shown to have various effects on immune function, although these effects are dependent on dose, animal model, and immune cell tested. Since the increased risk for infection and tumor that is observed in the elderly is thought to be in part secondary to waning cell-mediated immunity, we investigated the effect of age on beta-endorphin immunomodulation of T-cell proliferation in a murine model. Spleen cells obtained from young and old BALB/c mice were cultured in vitro with various mitogens with and without beta-endorphin. beta-Endorphin at 10(-8) M on day 3 of culture significantly enhanced concanavalin A (2.0 micrograms/10(6) cells per ml) mitogenesis but not phytohemagglutinin or lipopolysaccharide mitogenesis. Moreover, this enhancement was shown only in spleen cells from young mice and was not blocked by the opiate receptor antagonist naloxone, which suggests that enhancement of mitogenesis by beta-endorphin was mediated by a non-opiate receptor. Finally, our results support an altered response to neuroimmunomodulation with age.     

Morphological study and comprehensive cellular constituents of milky spots in the human omentum

Objective: To analyze morphological features of omental milky spots (MS). Method: Hematoxylin-eosin staining and immunohistochemistry technique were used to study the omental MS of gastric cancer (GC) patients and rectal cancer (RC) patients. We focused on morphological features of MS and conducted quantitative analysis on the cells number and cellular constituents. Differences in MS parameters between GC and RC were also analyzed. Results: Various shapes of MS were mainly round, oval, irregular form in the adipose and perivascular annular. The median MS perimeter was 2752 (range 817~7753) computer-based pixels. The median value of immune cells in one MS was 141 (43~650), comprising T lymphocytes (46.1%), B lymphocytes (28.4%), macrophages (12.4%) and other immune cells (13.1%). Relatively high density of vessels in MS could be calculated by micro-vessel density (MVD) as 4 (0~13). The median value of mesothelial cells loosely arranged in the surface layer was 5 (0~51). There were no significant differences in MS perimeter, MVD, the number of mesothelial cells, total immune cells, T lymphocytes and macrophages between GC and RC (P>0.05), while the number of MS B lymphocytes in RC was significantly higher than that in GC (P<0.001). Conclusion: MS are primary immune tissues in the omentum and structural bases for development and progression of peritoneal dissemination of GC and RC. Analyzing the morphology and cellular constituents could help understanding the mechanism of peritoneal metastasis.     

Augmentation of host defense mechanisms against tumor by sperabillin polymers,new basic peptidyl biopolymers,in mice

Sperabillin polymers, which have been shown recently to have antitumor activity, are new basic peptidyl polymers composed of a pseudo-peptide antibiotic, sperabillin A. The polymers, HP-2 (MW 9990), AP-2 (MW 20,100) and AB-2 (MW 35,000), were found to potently activate murine peritoneal macrophages. The phagocytosis-dependent respiratory burst and Fcγ receptor expression of peritoneal macrophages from C57BL/6 mice were enhanced after in vitro cultivation with these polymers. When HP-2, a representative of these polymers, was intraperitoneally injected into mice, the number of peritoneal exudate cells increased and phagocytosis-dependent respiratory burst and class II (I-A) antigen expression of peritoneal macrophages were augmented. These macrophages showed strong inhibitory activity against the growth of murine tumor cell lines such as EL4 lymphoma and B16 melanoma. Nitrogen oxide, tumor necrosis factor (TNF) and interleukin 1 (IL-1) might be required for this inhibitory activity. Moreover, in mice treated with HP-2, splenocyte counts also increased and non-specific killer activity of the splenocytes was augmented. These results indicate that sperabillin polymers are new macrophage activators.     

Nodular histiocytic aggregates in the greater omentum of patients with ovarian cancer

Nodular histiocytic aggregate (NHA) of the omentum is a rare benign proliferative process composed predominantly of histiocytes with scattered mesothelial cells. NHA is a differential diagnosis for neoplasms or metastatic tumors in cancer patients. To further clarify this clinical pitfall issue, the authors investigated surgical samples of the greater omentum from 96 patients with gastrointestinal malignancies and 53 patients with gynecologic neoplasms. Visible NHA of greater omentum was identified in 3 patients with ovarian neoplasms (borderline mucinous cystadenoma, low-grade papillary serous cystadenocarcinoma, and juvenile granulosa-cell tumor) but in none of the patients with gastrointestinal malignancies. Similar lesion was also identified on the cell blocks from peritoneal washings in 1 of the 3 patients. Grossly, the lesions formed small yellow-red nodules on the greater omentum, and the NHA lesion was also found diffusely on the surface of the appendix and fallopian tubes in 2 of the 3 patients. Histological study showed that typical NHA changes over an inflammatory background, which may indicate that NHA is a consequence of a chronic inflammatory process of omentum. The predominant infiltration of T lymphocytes in the NHA lesions indicates that the aggregation of histiocytes may be related to the activation of T-cell immunity. This report has first demonstrated visible NHA in the greater omentum of patients with ovarian malignancies, and awareness of this entity should be brought to clinicians to avoid misdiagnosis.     

Local delivery of recombinant adenovirus expressing hepatitis B virus X protein and interleukin-12 results in antitumor effects via inhibition of hepatoma cell growth and intervention of tumor microenvironment

Hepatocellular carcinoma (HCC) is a typical hypervascular tumor. Our previous studies have demonstrated that hepatitis B virus X protein (HBx) was able to inhibit the growth of HCC cells via inducing apoptosis and inhibiting tumor angiogenesis. Interleukin-12 (IL-12) is a disulfide-linked heterodimeric cytokine with potent immunostimulatory activity and anti-angiogenic properties. In this study, to further investigate the regulatory effect of IL-12 on HBx-mediated intervention of hepatoma microenvironment especially on intervention of neovessels and immune microenvironment, we constructed the recombinant adenovirus expressing HBx and mouse IL-12 named Ad-HBx-mIL-12. HBx-mIL-12 could effectively suppress tumor growth and induce apoptosis in vivo. Moreover, treatment with Ad-HBx-mIL-12 not only induced a massive accumulation of immune cells (CD8(+) T leukocytes, macrophages and dendritic cells) in tumors in situ, also apparently reduced the number of angiogenic blood vessels within tumor tissues. These results suggest that HBx-mIL-12 can not only induce cell cycle arrest and apoptosis in HCC cells, but also effectively shift the tumor microenvironment from pro-oncogenic to antitumor through recruitment of immune cells and inhibiting stromal cell growth, such as vascular endothelial cells.     

The F‐prostaglandin receptor is a novel marker for tumor endothelial cells in renal cell carcinoma

Tumor angiogenesis is necessary for tumor progression and metastasis; therefore, tumor blood vessels are potential therapeutic targets in anticancer therapy. We previously reported that tumor endothelial cells (TECs) exhibit different phenotypes compared with normal endothelial cells (NECs), and microarray analyses of mouse TECs and NECs have shown that several genes are upregulated in TECs compared with NECs. Among these genes, the expression levels of prostaglandin F receptor (PTGFR) mRNA, which encodes the prostaglandin F receptor (FP), were higher in TECs than in NECs. It has been reported that FP and its ligand, prostaglandin F_2α, are involved in tumor angiogenesis. However, there have been no reports of the expression of PTGFR in the tumor vessels of renal cell carcinoma (RCC). Thus, we isolated human TECs (hTECs) from RCCs. The expression levels of PTGFR mRNA were also upregulated in hTECs. In addition, immunostaining showed that the PTGFR was expressed in human tumor blood vessels in vivo. These findings suggested that PTGFR is a novel TEC marker and that it may be a novel target for antiangiogenic therapy for RCC.     

Inhibitory receptors as targets for cancer immunotherapy

Inhibitory receptors expressed on T cells control immune responses while limiting autoimmunity. However, tumors can hijack these “checkpoints” for protection from immune attack. Tumor‐specific T cells that exhibit an exhausted, unresponsive phenotype express high levels of inhibitory receptors including CTLA4, PD1, and LAG3, among others. Intratumoral regulatory T cells promote immunosuppression and also express multiple inhibitory receptors. Overcoming this inhibitory receptor‐mediated immune tolerance has thus been a major focus of recent cancer immunotherapeutic developments. Here, we review how boosting the host's immune system by blocking inhibitory receptor signaling with antagonistic mAbs restores the capacity of T cells to drive durable antitumor immune responses. Clinical trials targeting the CTLA4 and PD1 pathways have shown durable effects in multiple tumor types. Many combinatorial therapies are currently being investigated with encouraging results that highlight enhanced antitumor immunogenicity and improved patient survival. Finally, we will discuss the ongoing identification and dissection of novel T‐cell inhibitory receptor pathways, which could lead to the development of new combinatorial therapeutic approaches.     

Tim-3 and its role in regulating anti-tumor immunity

Immunotherapy is being increasingly recognized as a key therapeutic modality to treat cancer and represents one of the most exciting treatments for the disease. Fighting cancer with immunotherapy has revolutionized treatment for some patients and therapies targeting the immune checkpoint molecules such as CTLA-4 and PD-1 have achieved durable responses in melanoma, renal cancer, Hodgkin's diseases and lung cancer. However, the success rate of these treatments has been low and a large number of cancers, including colorectal cancer remain largely refractory to CTLA-4 and PD-1 blockade. This has provided impetus to identify other co-inhibitory receptors that could be exploited to enhance response rates of current immunotherapeutic agents and achieve responses to the cancers that are refectory to immunotherapy. Tim-3 is a co-inhibitory receptor that is expressed on IFN-g-producing T cells, FoxP3+ Treg cells and innate immune cells (macrophages and dendritic cells) where it has been shown to suppress their responses upon interaction with their ligand(s). Tim-3 has gained prominence as a potential candidate for cancer immunotherapy, where it has been shown that in vivo blockade of Tim-3 with other check-point inhibitors enhances anti-tumor immunity and suppresses tumor growth in several preclinical tumor models. This review discusses the recent findings on Tim-3, the role it plays in regulating immune responses in different cell types and the rationale for targeting Tim-3 for effective cancer immunotherapy.