Carbonic anhydrase II in the endothelium of glial tumors: a potential target for therapy

Carbonic anhydrase isozyme II (CA II) is a cytosolic enzyme that is highly expressed in most organs, including the brain, where it is mainly located in the oligodendrocytes. Recent studies have shown that its expression is induced in the endothelium of neovessels in melanoma and esophageal, renal, and lung cancer. Immunological studies further indicate that CA II represents a major target antigen stimulating an autoantibody response in melanoma patients. These results prompted us to investigate endothelial CA II expression in two types of brain cancer: oligodendrogliomas and astrocytomas. A series of 255 astrocytoma and 71 oligodendroglial tumor specimens was immunostained for CA II. The staining results were correlated with a number of different clinicopathological factors and survival data. CA II showed weak or no expression in low-grade tumors, while grade 3 mixed oligoastrocytoma and glioblastoma multiforme were the most positively stained tumor types. Survival analysis indicated that endothelial CA II staining is significantly associated with a poor prognosis in patients with astrocytomas. About 17% of patients with CA II-negative tumors (weak or no endothelial signal) were still alive at the end of the follow-up period of five years. The presence of CA II in the tumor endothelium suggests that it may play an important functional role in tumor metabolism. From a clinical perspective, the results also open new avenues for selecting tumor types for dendritic cell therapy trials.     

Carbonic anhydrase IX (CA IX) mediates tumor cell interactions with microenvironment

Expression of CA IX is normally restricted to the mucosa of alimentary tract, but on the other hand, it takes place in a high percentage of human cancers derived from tissues which are normally CA IX-negative. It is a transmembrane protein with two extracellular domains: carbonic anhydrase (CA) with a high catalytic activity and a proteoglycan-like segment (PG), mediating cell-cell adhesion. Both CA and PG domains interact with the microenvironment and they could play a role in tumorigenesis, but their roles are poorly understood. The present work characterizes some newly recognized properties of the PG. One of them is a prevalently negative charge, caused by a high proportion of dicarboxylic amino acids. This is reflected by easy dissociation of complexes formed by PG either with monoclonal antibody M75 or with the cell surface receptor already at slightly acidic pH. This property might facilitate separation of cells from the primary tumor. Released cells may subsequently attach elsewhere in the organism and eventually start metastatic growth. Another aim of the present study was to identify human tumor cell lines which are expressing the presumed CA IX receptor molecule. The same cell lines were also tested for the presence of CA IX protein; we found that expression of CA IX and of the receptor is independent of each other. In addition, we examined the species specificity of CA IX receptors. The PG domain, which contains the epitope of mAb M75 -PGEEDLP- overlapping with the binding site for putative receptor is relatively conserved in evolution: human and rat CA IX cross-react with M75 antibody on western blots. Consistently with this, human and rat cells can attach to purified human CA IX protein. On the other hand, murine CA IX contains an entirely different equivalent of PG sequence and it does not react with M75 antibody or attach to human CA IX protein. This is suggestive of the co-evolution of CA IX protein together with its receptor.     

Carbonic anhydrase IX expression is associated with tumor progression and a poor prognosis of lung adenocarcinoma

Carbonic anhydrase (CA) IX catalyzes the hydration of carbon dioxide into carbonic acid and participates in a variety of physiological and biological processes. The aim of this study was to evaluate the prognostic significance of CA IX expression in patients with lung adenocarcinoma. Standard immunohistochemical techniques were used to study CA IX expression in 134 patients who underwent curative resection for adenocarcinoma of the lung at our hospital between January 1995 and December 1996. We evaluated the correlations between CA IX expression levels on cancer cells and clinicopathological factors. CA IX expression was not observed in normal lung tissue or specimens from non-invasive adenocarcinomas. CA IX immunostaining was detected in 33 (24.6%) invasive adenocarcinoma cases. Poor differentiated histological phenotype (p=0.0015), pathological stage (p=0.0400), vascular invasion (p=0.0009) and lymphatic permeation (p=0.0050) were significantly related to CA IX expression. On univariate analysis, CA IX positive cases showed significantly shorter overall survival (p=0.0083) and disease-free survival (p=0.0122). In particular, the overall and disease-free survivals in stages I+II were significantly shorter in the CA IX positive than in the CA IX negative cases (p=0.0269 and 0.0011, respectively). Our results suggest that CA IX expression is strongly associated with tumor progression and indicates a poor prognosis for patients with stages I+II lung adenocarcinoma.     

Carbonic anhydrase IX in early-stage non-small cell lung cancer.

PURPOSE: Tumor hypoxia is associated with poor prognosis and increased tumor aggressiveness. Carbonic anhydrase (CA) IX, an endogenous marker for tumor hypoxia, catalyzes the hydration of carbon dioxide into carbonic acid and contributes to the pH regulation of tumor cells. Therefore, CA IX might allow tumors to acclimate to a hypoxic microenvironment, promoting tumor cell proliferation. We hypothesized that CA IX expression is related to tumor cell proliferation and poor disease-free survival in patients with early-stage non-small-cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: CA IX expression was measured in 75 resected NSCLC tumors to assess prognostic implications for disease-free survival. The relationship of CA IX expression with microvessel density (MVD) and proliferation (Ki-67) index was assessed via colocalization analysis. RESULTS: All patients had operable NSCLC (stage I, 58; stage II, 17). CA IX expression was present in 54 (72%) of 75 patients and was associated with tumor necrosis (P < 0.05). CA IX-positive tumor areas showed greater cell proliferation as measured by Ki-67 index (P < 0.05) and less MVD (P < 0.05) than did CA IX-negative areas in colocalization analysis. The percentage of CA IX-positive tumor cells was significantly related to postoperative recurrence and poor disease-free survival (P < 0.05). Ki-67 index and pathologic stage were also independent prognostic factors for worse disease-free survival (P < 0.05). CONCLUSIONS: CA IX expression of tumor cells may be an indicator for poor disease-free survival in early-stage NSCLC.     

Carbonic anhydrase IX is an independent predictor of survival in advanced renal clear cell carcinoma: implications for prognosis and therapy.

PURPOSE: Metastatic renal cell carcinoma (RCC) has a poor prognosis and an unpredictable course. To date, there are no molecular markers which can reliably predict RCC outcome. We investigated whether a novel kidney cancer marker, carbonic anhydrase IX (CAIX), is associated with progression and survival. EXPERIMENTAL DESIGN: Immunohistochemical analysis using a CAIX monoclonal antibody was performed on tissue microarrays constructed from paraffin-embedded specimens from patients (N = 321) treated by nephrectomy for clear cell RCC. CAIX staining was correlated with response to treatment, clinical factors, pathologic features, and survival. RESULTS: CAIX staining was present in 94% of clear cell RCCs. Survival tree analysis determined that a cutoff of 85% CAIX staining provided the most accurate prediction of survival. Low CAIX (相似文献   

Carbonic anhydrase IX is highly expressed in hereditary nonpolyposis colorectal cancer.

Carbonic anhydrase (CA) II, CA IX, and CA XII are expressed in various neoplasias and have been linked to tumorigenesis. We examined their expression in three different groups of colorectal cancer [i.e., microsatellite stable (MSS), microsatellite instable (MSI), and hereditary nonpolyposis colorectal cancer (HNPCC)]. First, we analyzed gene expression profiles of 113 specimens by a microarray method to study the expression of various CA isozymes in the subgroups of colorectal cancer. The results indicated that mRNAs for CA II and CA XII are down-regulated and CA IX mRNA is up-regulated in all three tumor categories when compared with the normal tissue. The up-regulation of CA IX was greatest in the HNPCC group. For more information, 77 specimens were immunohistochemically stained to study the levels of CA II, CA IX, and CA XII. Immunohistochemical analyses further confirmed that the subgroups express CA II, CA IX, and CA XII differentially, and the HNPCC tumors express high levels of CA IX. Expression of these CAs did not correlate to Dukes stage or grade of differentiation. Our results show that CAs are differentially expressed in the subgroups of colorectal cancer, and CA IX expression seems to be very high in most cases of HNPCC. CA IX could be a potential diagnostic and therapeutic target in HNPCC.     

Efficient tumor antigen loading of dendritic antigen presenting cells by transimmunization

Extracorporeal photochemotherapy (ECP), or photopheresis, was originally introduced for the management of patients with cutaneous T cell lymphoma (CTCL). Today, ECP remains the only FDA approved tumor-targeting selective immunotherapy for the treatment of any cancer. The key cellular events permitting ECP-induced anti-tumor immunity against CTCL are the induction of apoptotis in the malignant T cells, and the induction of monocyte-to-dendritic cell (DC) differentiation. In standard ECP, leukocytes extracorporeally exposed to psoralen and ultraviolet A light (UVA) are circulated back to the patient. However, recent findings suggest that co-incubation of these cells prior to re-infusion allows for more efficient phagocytosis and processing of the apoptotic malignant T cells by the newly formed DCs. Moreover, such a co-incubation step permits the direct external manipulation of this system and the design of strategies to augment the production of tumor-loaded DCs. These considerations have led to the development of Transimmunization, so named because it causes transfer of tumor antigens to newly formed dendritic cells capable of initiating immunization against the tumor cells, as the replacement technology for ECP. We will review the scientific understanding of ECP and explain how this can lead a more efficient, potentially broadly applicable, immunotherapy for cancer.     

Carbonic anhydrase IX expression, a novel surrogate marker of tumor hypoxia, is associated with a poor prognosis in non-small-cell lung cancer.

PURPOSE: To evaluate carbonic anhydrase (CA) IX as a surrogate marker of hypoxia and investigate the prognostic significance of different patterns of expression in non-small-cell lung cancer (NSCLC). METHODS: Standard immunohistochemical techniques were used to study CA IX expression in 175 resected NSCLC tumors. CA IX expression was determined by Western blotting in A549 cell lines grown under normoxic and hypoxic conditions. Measurements from microvessels to CA IX positivity were obtained. RESULTS: CA IX immunostaining was detected in 81.8% of patients. Membranous (m) (P =.005), cytoplasmic (c) (P =.018), and stromal (P <.001) CA IX expression correlated with the extent of tumor necrosis (TN). The mean distance from vascular endothelium to the start of tumor cell positivity was 90 micro m, which equates to an oxygen pressure of 5.77 mmHg. The distance to blood vessels from individual tumor cells or tumor cell clusters was greater if they expressed mCA IX than if they did not (P <.001). Hypoxic exposure of A549 cells for 16 hours enhanced CA IX expression in the nuclear and cytosolic extracts. Perinuclear (p) CA IX (P =.035) was associated with a poor prognosis. In multivariate analysis, pCA IX (P =.004), stage (P =.001), platelet count (P =.011), sex (P =.027), and TN (P =.035) were independent poor prognostic factors. CONCLUSION: These results add weight to the contention that mCA IX is a marker of tumor cell hypoxia. The absence of CA IX staining close to microvessels suggests that these vessels are functionally active. pCA IX expression is representative of an aggressive phenotype.     

Carbonic anhydrase XII is a marker of good prognosis in invasive breast carcinoma

Hypoxia and pH influence gene expression in tumours, and it is becoming increasingly clear that the pattern of genes expressed by a tumour determines its growth and survival characteristics. Hypoxia-inducible factor-1 (HIF-1) is a key mediator of the cellular response to hypoxia and high HIF-1 expression has been identified as a poor prognostic factor in tumours. Recently, we identified the tumour-associated carbonic anhydrases (CA), CA9 and CA12 as hypoxia-inducible in tumour cell lines. Furthermore, we identified CA IX to be a poor prognostic factor in breast cancer. The aim of this study was to assess the prognostic significance of CA XII. CA XII expression was studied by immunohistochemistry in a series of 103 cases of invasive breast cancer and any association with recognised prognostic factors or relation with the outcome was examined. CA XII expression was present in 77 out of 103 (75%) cases and was associated with lower grade (P=0.001), positive estrogen receptor status (P<0.001), and negative epidermal growth factor receptor status (P<0.001). Furthermore, although CA XII expression was associated with an absence of necrosis (P<0.001), expression of CA XII in some high-grade tumours was induced in regions directly adjacent to morphological necrosis. Additionally, using univariate analysis, CA XII positive tumours were associated with a lower relapse rate (P=0.04) and a better overall survival (P=0.01). In conclusion, CA XII expression is influenced both by factors related to differentiation and hypoxia in breast cancer in vivo and CA XII expression is associated with a better prognosis in an unselected series of invasive breast carcinoma patients.     

Carbonic anhydrase IX expression predicts outcome of interleukin 2 therapy for renal cancer.

PURPOSE: Renal cancer response to interleukin 2 (IL-2) therapy and patient survival has been correlated with tumor histology and carbonic anhydrase IX (CAIX) expression. In an effort to confirm and expand these observations, we examined CAIX expression in pathology specimens from renal cancer patients who had previously received IL-2 therapy. EXPERIMENTAL DESIGN: Paraffin-embedded tissue sections of renal cancer were immunostained with the MN-75 monoclonal antibody to CAIX and expression levels were correlated with histologic findings and clinical outcome. RESULTS: Tissue specimens were obtained from 66 patients; 27 of whom (41%) had responded to IL-2-based therapy. Fifty-eight specimens were assessed as clear cell, with 56, 33, and 4 having alveolar, granular, and papillary features, respectively. Twenty-four (36%), 31 (47%), and 11 (17%) were classified into good, intermediate, and poor prognosis groups according to the Upton pathology model. Forty-one specimens (62%) had high CAIX expression. Twenty-one of 27 (78%) responding patients had high CAIX expressing tumors compared with 20 of 39 (51%) nonresponders (odds ratio, 3.3; P = 0.04). Median survival was prolonged (P = 0.04) and survival >5 years was only seen in high CAIX expressers. In patients with intermediate pathologic prognosis, all nine responders had high CAIX expression versus 11 of 22 nonresponders. A resultant group with good pathologic prognosis alone or with intermediate pathologic prognosis and high CAIX contained 26 of 27 (96%) responders compared with 18 of 39 (46%) nonresponders (odds ratio, 30; P < 0.01) and exhibited longer median survival (P < 0.01). CONCLUSIONS: CAIX expression seems to be an important predictor of outcome in renal cell carcinoma patients receiving IL-2-based therapy and may enhance prognostic information obtained from pathology specimens.     

Carbonic anhydrase 2 is a novel invasion‐associated factor in urinary bladder cancers

Rat bladder cancer is nearly always papillary non‐invasive urothelial carcinoma (UC). To establish an animal model mimicking invasive UC that arises from papillary non‐invasive UC in the bladder, male human c‐Ha‐ras proto‐oncogene transgenic rats (Hras128) were treated with 0.05% N‐butyl‐N‐(hydroxybutyl)nitrosameine (BBN) in their drinking water and/or 0.1% phenylethyl isothiocyanate (PEITC) in their diet as follows: BBN (8 weeks)→PEITC (8 weeks); PEITC (8 weeks)→BBN (8 weeks); BBN alone (16 weeks); PEITC alone (16 weeks); and no treatment. At the end of week 16, the highest incidence of invasive UC was observed in the BBN→PEITC group. Therefore, we used Hras128 rats treated with BBN followed by PEITC as a model of invasive bladder cancer to identify invasion‐associated proteins. Proteome analysis was performed to compare the protein profiles of invasive and non‐invasive UC in Hras128 rats. We identified 49 proteins that were either overexpressed or underexpressed in invasive UC but not in non‐invasive UC. Immunohistochemical analysis of carbonic anhydrase 2 (CA2), an overexpressed protein, showed that the relative number of CA2‐positive UC was significantly higher for invasive UC compared to non‐invasive UC in rats. Moreover, the incidence of CA2‐positive cancers was also significantly higher for human muscle‐invasive bladder cancer (MIBC) compared to non‐MIBC (NMIBC) and was positively associated with the progression of NMIBC. Our findings indicate that CA2 is an invasion‐associated factor and suggest that it could serve as a potential therapeutic molecular target for bladder cancers.     

Rapamycin-induced endothelial cell death and tumor vessel thrombosis potentiate cytotoxic therapy against pancreatic cancer.

PURPOSE: Despite current chemotherapies, pancreatic cancer remains an uncontrollable, rapidly progressive disease. Here, we tested an approach combining a recently described antiangiogenic drug, rapamycin, with standard gemcitabine cytotoxic therapy on human pancreatic tumor growth. EXPERIMENTAL DESIGN: Tumor growth was assessed in rapamycin and gemcitabine-treated nude mice orthotopically injected with metastatic L3.6pl human pancreatic cancer cells. H&E staining was performed on tumors, along with Ki67 staining for cell proliferation and immunohistochemical terminal deoxynucleotidyl transferase-mediated nick end labeling and CD31 analysis. Rapamycin-treated tumor vessels were also directly examined in dorsal skin-fold chambers for blood flow after thrombosis induction. Cell death in human umbilical vein endothelial cells was assessed by flow cytometry after annexin-V staining. RESULTS: Rapamycin therapy alone inhibited tumor growth and metastasis more than gemcitabine, with remarkable long-term tumor control when the drugs were combined. Mechanistically, H&E analysis revealed tumor vessel endothelium damage and thrombosis with rapamycin treatment. Indeed, dorsal skin-fold chamber analysis of rapamycin-treated tumors showed an increased susceptibility of tumor-specific vessels to thrombosis. Furthermore, terminal deoxynucleotidyl transferase-mediated nick end labeling/CD31 double staining of orthotopic tumors demonstrated apoptotic endothelial cells with rapamycin treatment, which also occurred with human umbilical vein endothelial cells in vitro. In contrast, gemcitabine was not antiangiogenic and, despite its known cytotoxicity, did not reduce proliferation in orthotopic tumors; nevertheless, rapamycin did reduce tumor proliferation. CONCLUSIONS: Our data suggest a novel mechanism whereby rapamycin targets pancreatic tumor endothelium for destruction and thrombosis. We propose that rapamycin-based vascular targeting not only reduces tumor vascularization, it decreases the number of proliferating tumor cells to be destroyed by gemcitabine, thus introducing a new, clinically feasible strategy against pancreatic cancer.     

热休克肿瘤细胞抗原负载的树突状细胞对结肠癌小鼠的治疗作用

背景与目的:目前通过树突状细胞诱导机体抗肿瘤免疫已经成为肿瘤免疫治疗的一种重要途径,热休克处理肿瘤细胞可提高肿瘤细胞抗原负载的树突状细胞分化成熟,增强其体内抗肿瘤作用.本实验研究热休克肿瘤细胞抗原负载的骨髓来源的树突状细胞(dendritic cell,DC)对结肠癌小鼠的治疗作用.方法:20只小鼠随分为4组,将小鼠结肠癌细胞CT26热休克处理后超声破膜,以其细胞裂解液负载BALB/c小鼠骨髓来源的DC,观察DC诱导肿瘤特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)杀伤活性;并将DC接种于荷瘤小鼠皮下,观察其对肿瘤生长的抑制作用及对荷瘤小鼠生存期的影响.结果:与冻融抗原-DC组、DC组、PBS组相比,热休克抗原-DC诱导的CTL对CT26肿瘤细胞具有显著的杀伤作用,相同效靶比(P=0.00)下其肿瘤特异性细胞毒活性强于前者{(0.99±0.19)g vs.[(1.27±0.28)g、(2.19±0.35)g、(2.14±0.27)g],P均=0.00};用其进行免疫后对小鼠肿瘤的生长具有显著的抑制作用[(128±18)mm3 vs. (313±52)mm3,P=0.04],并能显著延长荷瘤小鼠的生存时间{(57±7)d vs. [(40±3)d、(25±3)d、(24±3)d],P均=0.00}.结论:热休克肿瘤细胞抗原负载的树突状细胞对结肠癌小鼠具有显著的治疗效果.     

LABAZ1: A metastatic tumor model for renal cell carcinoma expressing the carbonic anhydrase type 9 tumor antigen

A metastatic renal cell carcinoma (RCC) tumor model xenograft that expresses the targetable, membrane-bound tumor-associated antigen carbonic anhydrase type 9 (CA IX) is described. The xenograft, established from a high-grade type-2 chromophil RCC (cRCC), has been serially transplanted in immune compromised mice, in which it grows orthotopically under the renal capsule, doubling its size every 9 weeks and sending metastases to the lung and liver at approximately 20 weeks. Tumors were capable of being imaged using a micro-PET (micro-positron emission tomograph) with an 18-fluorodeoxyglucose (18-FDG) tracer. Subsequent xenograft generations have conserved immunohistochemical and ultrastructural properties typical for malignant renal epithelium-derived neoplasia (vimentin+, CK-19+, CA IX+ with hypoxia-inducible factor (HIF)-1 alpha constitutive expression) and have demonstrated extensive proliferation, lack of apoptosis, severe genetic alterations, and molecular expression alterations; transforming growth factor beta 1 (TGF-beta 1), hepatocyte growth factor (HGF), proto-oncogene (c-met), matrix metalloproteinase (MMP)-1, and vascular endothelial growth factor (VEGF) C and D were overexpressed, whereas human epidermal growth factor receptor (HER)-2, MMP-2 and MMP-9, VEGF-R3, p53, and p27 were severely down-regulated, suggesting a proangiogenic environment, local invasiveness, and facilitated lymphatic metastasis. Altogether, LABAZ1 provides a relevant and flexible model to study the biology of cRCC, the role of CA IX in RCC tumorigenesis, progression, and metastasis, and a platform for testing new targeted therapeutic strategies.     

Carbonic anhydrase IX as a marker for poor prognosis in soft tissue sarcoma.

PURPOSE: Hypoxia is associated with malignant progression and poor outcome in several human tumors, including soft tissue sarcoma. Recent studies have suggested that carbonic anhydrase (CA) IX is an intrinsic marker of hypoxia, and that CA IX correlates with poor prognosis in several types of carcinoma. The aim of this study was to quantify the extent of CA IX expression and to investigate whether CA IX is a marker for poor prognosis in soft tissue sarcoma patients at high risk of developing metastasis. EXPERIMENTAL DESIGN: Archival paraffin-embedded blocks were retrieved from 47 patients with deep, large, high-grade soft tissue sarcoma. Sections from two separate and representative tumor areas were immunostained for CA IX, and the CA IX-positive area fraction was quantified by image analysis, excluding areas of normal stroma and necrosis that were identified from serial H&E-stained sections. Patients were then subject to survival analysis. RESULTS: CA IX-positive area fractions of viable tumor tissue varied significantly between tumors (range, 0-0.23; median, 0.004), with positive membranous CA IX staining in 66% (31 of 47) of the tumors. Patients with CA IX-positive tumors had a significantly lower disease-specific and overall survival than patients with CA IX-negative tumors (P = 0.033 and P = 0.044, respectively). CONCLUSIONS: These data suggest that CA IX, a potential intrinsic marker of hypoxia, predicts for poor prognosis in patients with deep, large, high-grade soft tissue sarcoma. Larger studies are required to determine whether CA IX has independent prognostic value in this group of tumors.     

树突状细胞疫苗肿瘤抗原负载方式的临床转化研究进展

树突状细胞（dendritic cell, DC）是体内功能最强的抗原提呈细胞,能将肿瘤抗原提呈给T细胞,是抗肿瘤免疫的启动者。DC疫苗的肿瘤免疫治疗虽已取得丰硕成果,但在其临床转化过程中还有很多问题需要解决,选择何种抗原负载方式则是其中之一。目前临床试验中常用的有肿瘤细胞裂解物负载DC、重组肿瘤相关抗原负载DC及携带肿瘤相关抗原信息的mRNA转染DC三种方法。本文对此三种方法在临床试验中的应用做一综述,为树突状细胞疫苗的临床应用提供参考。     

肿瘤诱导树突状细胞凋亡研究进展

树突状细胞作为最重要的抗原递呈细胞,在抗肿瘤免疫中起重要作用.恶性肿瘤可以通过分泌细胞因子、蛋白质、神经酰胺和神经节苷脂引起树突状细胞功能缺陷及数量减少,还可以诱导树突状细胞发生凋亡.因此,肿瘤相关抗原不能有效传递给T细胞,激活抗肿瘤免疫应答.利用某些蛋白和细胞因子对树突状细胞进行预处理,可以减少肿瘤诱导凋亡的发生,基因技术可以增强树突状细胞耐受凋亡的能力.现综述恶性肿瘤诱导树突状细胞凋亡的研究进展.