Proportional Upregulation of CD97 Isoforms in Glioblastoma and Glioblastoma-Derived Brain Tumor Initiating Cells

CD97 is a novel glioma antigen that confers an invasive phenotype and poor survival in patients with glioblastoma (GBM), the most aggressive primary malignant brain tumor. The short isoform of CD97, known as EGF(1,2,5), has been shown to promote invasion and metastasis, but its role in gliomas and GBM-derived brain tumor initiating cells (BTICs) has not been studied. We sought to characterize CD97 expression among gliomas and identify the specific isoforms expressed. The short isoform of CD97 was identified in GBM and GBM-derived BTICs, but not low grade or anaplastic astrocytomas. All samples expressing the EGF(1,2,5) isoform were also found to express the EGF(1,2,3,5) isoform. These isoforms are believed to possess similar ligand binding patterns and interact with chondroitin sulfate, a component of the extracellular matrix, and the integrin α5β1. Using data acquired from the Cancer Genome Atlas (TCGA), we show that CD97 is upregulated among the classical and mesenchymal subtypes of GBM and significantly decreased among IDH1 mutant GBMs. Given its proven roles in tumor invasion, expression among aggressive genetic subtypes of GBM, and association with overall survival, CD97 is an attractive therapeutic target for patients with GBM.     

Changes in cytoskeleton-associated molecules in cells with different degrees of proliferation and metastatic ability

An antiserum prepared against the Triton-insoluble cytoskeleton of in vivo grown B16 melanoma tumor has been used to analyze the differential expression of cytoskeleton-associated molecules in cells with different degrees of proliferation and metastatic ability. This antiserum identified a major 97 kd molecule associated with the cytoskeletal fraction in B16 melanoma tumors, mouse embryo and in proliferating lymphocytes, with no reactivity with the 97 kd species in non proliferating lymphocytes. The antiserum revealed immune reactivity with a 180 kd Triton-insoluble species in normal adult mouse liver and kidney. A comparison of tumor cells with differing metastatic ability also showed a minor 180 kd component in poorly metastatic cells which appeared decreased and partly degraded in its more invasive counterpart. The differential recognition of a 97 kd species in resting and proliferating lymphocytes, as well as the different cleavage of a 180 kd species in tumor cells of differing metastatic ability, implies a role for these molecules in cell proliferation. The fact that these differences can be detected with an antiserum to tumor cell cytoskeleton suggests that this Triton-insoluble fraction may be a good source of molecules involved in growth control.     

Silencing of CD44 Gene Expression in Human 143-B Osteosarcoma Cells Promotes Metastasis of Intratibial Tumors in SCID Mice

Osteosarcoma (OS) is the most frequent primary malignant bone cancer in children and adolescents with a high propensity for lung metastasis. Therefore, it is of great importance to identify molecular markers leading to increased metastatic potential in order to devise more effective therapeutic strategies that suppress metastasis, the major cause of death in OS. CD44, the principal receptor for the extracellular matrix component hyaluronan (HA), is frequently found overexpressed in tumor cells and has been implicated in metastatic spread in various cancer types. Here, we investigated the effects of stable shRNA-mediated silencing of CD44 gene products on in vitro and in vivo metastatic properties of the highly metastatic human 143-B OS cell line. In vitro, CD44 knockdown resulted in a 73% decrease in the adhesion to HA, a 57% decrease in the migration rate in a trans-filter migration assay, and a 28% decrease in the cells'' capacity for anchorage-independent growth in soft agar compared to the control cells, implicating that CD44 expression contributes to the metastatic activity of 143-B cells. However, making use of an orthotopic xenograft OS mouse model, we demonstrated that reduced CD44 expression facilitated primary tumor growth and formation of pulmonary metastases. The enhanced malignant phenotype was associated with decreased adhesion to HA and reduced expression of the tumor suppressor merlin in vivo. In conclusion, our study identified CD44 as a metastasis suppressor in this particular experimental OS model.     

荧光素酶标记人胃癌细胞裸鼠原位移植模型的建立

目的建立荧光素酶标记人胃癌原位异种移植模型。方法将萤火虫荧光素酶作为标记基因导入人胃癌MGC803细胞,建立稳定表达荧光素酶的细胞,将其接种裸鼠胃壁浆膜下,建立胃癌裸鼠原位肿瘤模型。用活体荧光成像系统检测肿瘤的发生发展,并进行小动物超声影像和病理学分析。结果裸鼠原位成瘤率为100%,活体荧光成像观察发现在接种第7天,就可以观察到肿瘤发光。21 d后肿瘤进入对数生长期,28 d后肿瘤出现明显坏死,平均荧光光子数呈现下降趋势。超声成像发现小鼠胃部有直径为8.39 mm,面积为28.92 mm2瘤块。结论荧光素酶标记可以实时监测原位异种移植人胃癌生长状况。     

CD1d-expressing breast cancer cells modulate NKT cell-mediated antitumor immunity in a murine model of breast cancer metastasis

Background

Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.

Methodology/Principal Findings

In this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors.

Conclusions/Significance

The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.     

A novel vascular-targeting peptide for gastric cancer delivers low-dose TNFα to normalize the blood vessels and improve the anti-cancer efficiency of 5-fluorouracil

Various vascular-targeted agents fused with tumor necrosis factor α (TNFα) have been shown to improve drug absorption into tumor tissues and enhance tumor vascular function. TCP-1 is a peptide selected through in vivo phage library biopanning against a mouse orthotopic colorectal cancer model and is a promising agent for drug delivery. This study further investigated the targeting ability of TCP-1 phage and peptide to blood vessels in an orthotopic gastric cancer model in mice and assessed the synergistic anti-cancer effect of 5-fluorouracil (5-FU) with subnanogram TNFα targeted delivered by TCP-1 peptide. In vivo phage targeting assay and in vivo colocalization analysis were carried out to test the targeting ability of TCP-1 phage/peptide. A targeted therapy for improvement of the therapeutic efficacy of 5-FU and vascular function was performed through administration of TCP-1/TNFα fusion protein in this model. TCP-1 phage exhibited strong homing ability to the orthotopic gastric cancer after phage injection. Immunohistochemical staining suggested that and TCP-1 phage/TCP-1 peptide could colocalize with tumor vascular endothelial cells. TCP-1/TNFα combined with 5-FU was found to synergistically inhibit tumor growth, induce apoptosis and reduce cell proliferation without evident toxicity. Simultaneously, subnanogram TCP-1/TNFα treatment normalized tumor blood vessels. Targeted delivery of low-dose TNFα by TCP-1 peptide can potentially modulate the vascular function of gastric cancer and increase the drug delivery of chemotherapeutic drugs.     

优化胶粘贴法建立裸鼠胃癌原位种植模型

目的通过对两种胶粘贴法建立的胃癌原位种植动物模型的比较研究,为探讨胃癌的发病机制和实验治疗提供理想的动物模型。方法用OB胶和FS生物蛋白胶法分别建立胃癌原位种植动物模型,观察和比较两种方法所建立的模型肿瘤生长状况、转移情况和形态学变化。结果FS生物蛋白胶组未出现肿瘤大片坏死,腹水形成率为85.7%,幽门梗阻发生率为57.1%;而OB胶组肿瘤大片坏死发生率为100%,腹水形成率为14.3%,未出现幽门梗阻。FS生物蛋白胶组有三例出现了肺和脑转移。结论FS生物蛋白胶法建立的裸鼠胃癌原位种植动物模型能更好的模拟人胃癌患者的临床过程,为研究人胃癌转移机制和实验治疗提供理想的动物模型。     

Behavior of metastatic and nonmetastatic breast tumors in old mice

Breast cancer incidence and mortality increase with age. A better understanding of the biological behavior of metastatic and nonmetastatic breast tumors in older subjects may help to develop improved breast cancer therapies. In this study, we used syngeneic metastatic (4TO7cg) and nonmetastatic (64pT) mouse breast tumor models at three age levels to evaluate various characteristics that are considered to be important for effective anti-breast cancer immunotherapy. These included tumor size and growth, metastases, vascularization, gene expression levels of the tumor-associated antigen (TAA) Mage-b (homologous to human MAGE-B) in primary breast tumors and metastases, and the presence of CD4(+) and CD8(+) T cells in the inguinal lymph nodes at the site of the tumor. The primary breast tumors and metastases were generated by injection of mouse mammary tumor cell lines 4TO7cg or 64pT into a mammary fat pad of normal 3-, 9-, or 21/24-month old BALB/c mice. In the nonmetastatic breast tumor model, significantly smaller tumors were observed in old compared with young mice. This was associated with a significant increase in the percentage of CD8(+) T cells in inguinal lymph nodes and significantly higher Mage-b expression levels in the primary tumors at old age. In the metastatic (4TO7cg) breast tumor model, a less pronounced, not statistically significant, smaller tumor size was found in the old mice, without a difference in the percentage of CD8(+) T cells or Mage-b expression levels. However, in this mouse model almost all metastases showed high levels of Mage-b expression (2- to 3-fold higher than the primary tumors in the same animals) regardless of age. These results indicate that the metastatic and nonmetastatic breast tumor models could be useful model systems to analyze how breast cancer vaccines for humans can be tailored to old age.     

Tumor‐propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis

Metastasis is the leading cause of morbidity for lung cancer patients. Here we demonstrate that murine tumor propagating cells (TPCs) with the markers Sca1 and CD24 are enriched for metastatic potential in orthotopic transplantation assays. CD24 knockdown decreased the metastatic potential of lung cancer cell lines resembling TPCs. In lung cancer patient data sets, metastatic spread and patient survival could be stratified with a murine lung TPC gene signature. The TPC signature was enriched for genes in the Hippo signaling pathway. Knockdown of the Hippo mediators Yap1 or Taz decreased in vitro cellular migration and transplantation of metastatic disease. Furthermore, constitutively active Yap was sufficient to drive lung tumor progression in vivo. These results demonstrate functional roles for two different pathways, CD24‐dependent and Yap/Taz‐dependent pathways, in lung tumor propagation and metastasis. This study demonstrates the utility of TPCs for identifying molecules contributing to metastatic lung cancer, potentially enabling the therapeutic targeting of this devastating disease.     

Synergistic anti-tumor responses after administration of agonistic antibodies to CD40 and IL-2: coordination of dendritic and CD8+ cell responses

In cancer, the coordinate engagement of professional APC and Ag-specific cell-mediated effector cells may be vital for the induction of effective antitumor responses. We speculated that the enhanced differentiation and function of dendritic cells through CD40 engagement combined with IL-2 administration to stimulate T cell expansion would act coordinately to enhance the adaptive immune response against cancer. In mice bearing orthotopic metastatic renal cell carcinoma, only the combination of an agonist Ab to CD40 and IL-2, but neither agent administered alone, induced complete regression of metastatic tumor and specific immunity to subsequent rechallenge in the majority of treated mice. The combination of anti-CD40 and IL-2 resulted in significant increases in dendritic cell and CD8(+) T cell number in advanced tumor-bearing mice compared with either agent administered singly. The antitumor effects of anti-CD40 and IL-2 were found to be dependent on CD8(+) T cells, IFN-gamma, IL-12 p40, and Fas ligand. CD40 stimulation and IL-2 may therefore be of use to promote antitumor responses in advanced metastatic cancer.     

Basement Membrane-Rich Organoids with Functional Human Blood Vessels Are Permissive Niches for Human Breast Cancer Metastasis

Metastasic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic intervention.     

CD169-positive macrophages enhance abscopal effect of radiofrequency ablation therapy in liver cancer

Radiofrequency ablation (RFA) is a widely used and effective treatment for primary or metastatic liver cancer with small-size lesions. However, the therapeutic effectiveness of RFA in controlling metastatic lesion or recurrence is still limited. As the major cell population in tumor microenvironment (TME), macrophages have been reported to be recruited to RFA-treated lesion, but their roles are still unclear. Herein, we successfully established the mouse model mimicking RFA-induced abscopal effect, in which RFA eliminated the local orthotopic liver tumor but failed to control growth of distant tumor. Correspondently, RFA suppressed protumoral activation of local tumor-associated macrophages (TAMs), but failed to reprogram TAMs in distance. Importantly, although RFA led to reduced proportion of hepatic CD169⁺ macrophages in local and decreased expression of immune inhibitory molecules Tim-3 and PD-L1, these alterations were not observed for CD169⁺ macrophages in distant TME. Further RNA-seq and flow cytometry analysis showed that hepatic CD169⁺ macrophages contributed to reprograming TME through recruiting CD8⁺ T/NK cells and suppressing accumulation of MDSCs/Tregs. Consistently, depletion of CD169⁺ macrophages in CD169-DTR mouse greatly promoted liver tumor progression and largely dampened RFA-induced tumor suppression. Notably, transfer of CD169⁺ macrophages synergistically enhanced RFA-induced inhibition of distant tumor. To our knowledge, this is the first study which demonstrates hepatic CD169⁺ macrophages as a key factor responsible for RFA-induced abscopal effect. Our data suggest RFA with transfer of CD169⁺ macrophages as a promising combination therapy to lessen metastasis or recurrence of liver cancer in patients.     

顺铂联合VEGF抗体对食管癌移植瘤小鼠免疫调节以及癌细胞增殖和肺转移的影响

摘要 目的：研究顺铂联合血管内皮生长因子（vascular endothelial growth factor,VEGF）治疗对食管癌移植瘤小鼠免疫功能、癌细胞增殖以及肺转移的影响。方法：30只BALB/c小鼠通过皮下注射食管癌移植瘤模型。一周后,30只食管癌移植瘤模型小鼠被随机均分为3组,即模型组、顺铂组和联合组。模型组不进行治疗,顺铂组腹腔注顺铂治疗,联合组腹腔注射顺铂联合尾静脉注射VEGF抗体进行治疗,共治疗7周。比较各组小鼠体重,食管癌移植瘤体积和重量,卵巢癌细胞肺组织转移结节数、癌细胞转移面积和转移病灶总数,以及食管癌移植瘤外周血CD4⁺、CD8⁺以及CD4⁺/CD8⁺ T淋巴细胞比例。结果：（1）顺博组和联合组小鼠体重均显著高于对照组,而联合组小鼠体重显著高于顺铂组（P<0.05）;（2）顺铂组和联合组小鼠CD4+和CD8+细胞比例均显著低于对照组（P<0.05）,而CD4⁺/CD8⁺却显著高于对照组（P<0.05）;（3）联合组小鼠CD4⁺和CD8⁺细胞比例均显著高于对照组（P<0.05）,而CD4⁺/CD8⁺却显著低于顺铂组（P<0.05）;（4）顺铂组和联合组小鼠食管癌肿瘤组织体积和重量,肺转移结节数、转移面积和转移病灶数均显著低于对照组（P<0.05）,而联合组小鼠显著低于顺铂组（P<0.05）。结论：VEGF抗体可以显著增强顺铂在体内对食管癌的抗癌特性,并有助于增强食管癌移植瘤小鼠免疫功能、抑制癌细胞体内增殖和肺部转移。     

Isolation and characterization of a metastatic hybrid cell line generated by ER negative and ER positive breast cancer cells in mouse bone marrow

Background

The origin and the contribution of breast tumor heterogeneity to its progression are not clear. We investigated the effect of a growing orthotopic tumor formed by an aggressive estrogen receptor (ER)-negative breast cancer cell line on the metastatic potential of a less aggressive ER-positive breast cancer cell line for the elucidation of how the presence of heterogeneous cancer cells might affect each other''s metastatic behavior.

Methods

ER positive ZR-75-1/GFP/puro cells, resistant to puromycin and non-tumorigenic/non-metastatic without exogenous estrogen supplementation, were injected intracardiacally into mice bearing growing orthotopic tumors, formed by ER negative MDA-MB-231/GFP/Neo cells resistant to G418. A variant cell line B6, containing both estrogen-dependent and -independent cells, were isolated from GFP expressing cells in the bone marrow and re-inoculated in nude mice to generate an estrogen-independent cell line B6TC.

Results

The presence of ER negative orthotopic tumors resulted in bone metastasis of ZR-75-1 without estrogen supplementation. The newly established B6TC cell line was tumorigenic without estrogen supplementation and resistant to both puromycin and G418 suggesting its origin from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. Compared to parental cells, B6TC cells were more metastatic to lung and bone after intracardiac inoculation. More significantly, B6TC mice also developed brain metastasis, which was not observed in the MDA-MB-231/GFP/Neo cell-inoculated mice. Low expression of ERα and CD24, and high expression of EMT-related markers such as Vimentin, CXCR4, and Integrin-β1 along with high CD44 and ALDH expression indicated stem cell-like characteristics of B6TC. Gene microarray analysis demonstrated a significantly different gene expression profile of B6TC in comparison to those of parental cell lines.

Conclusions

Spontaneous generation of the novel hybrid cell line B6TC, in a metastatic site with stem cell-like properties and propensity to metastasize to brain, suggest that cell fusion can contribute to tumor heterogeneity.     

中药组方黄芩汤抑制胃癌细胞增殖的实验研究

目的探讨黄芩汤对胃癌细胞生长增殖及干细胞标志物表达的影响。 方法采用体外优化培养的胃癌细胞株SGC-7901模型,分别加入生理盐水、5-FU、黄岑汤和5-FU+黄芪汤干预,MTT法检测胃癌细胞增殖情况,Real-time PCR检测胃癌干细胞相关表面标志物CD44、EpCAM、CD90的表达情况;并进一步利用胃癌细胞裸鼠荷瘤模型,观察黄岑汤协同5-FU对体内肿瘤生长的抑制效果。细胞增殖曲线多组间数据比较采用重复测量数据方差分析,单时间点多组数据采用单因素方差分析,组间比较采用Tukey法。 结果优化培养的胃癌细胞SGC- 7901呈球形生长,细胞活性染色显示良好的活性状态;MTT结果显示,黄岑汤组（0.44±0.04）能够抑制胃癌细胞的增殖,从第4天起与对照组（0.59±0.02）相比,差异具有统计学意义（F = 39.550,P < 0.01）;黄岑汤联合5-FU组（0.36±0.04）加强5-FU对肿瘤细胞的抑制作用,从第3天起与对照组（0.50±0.01）相比,差异具有统计学意义（F = 10.670,P < 0.01）,与5-FU组（0.42±0.03）对比,差异具有统计学意义（F = 10.670,P < 0.05）;Real-time PCR结果显示黄岑汤联合5-FU可抑制胃癌细胞中肿瘤干细胞相关表面标志物CD44、EpCAM、CD90表达（P < 0.01）;移植瘤生长抑制实验发现黄芪汤组对荷瘤鼠移植瘤的生长具有抑制作用,与对照组相比移植瘤体积减小,差异具有统计学意义（P < 0.01）,黄岑汤联合5-FU组与5-FU组相比对移植瘤抑制效果更明显,差异具有统计学意义（P < 0.01）。 结论黄芪汤能抑制胃癌细胞的生长和增殖,并能协同5-FU产生抗肿瘤作用。     

Orthotopic mouse model of colorectal cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.     

小鼠Lewis肺癌原位模型的建立

目的利用Matrigel与Lewis制备细胞混悬液注射于小鼠左肺内,建立小鼠Lewis肺癌原位模型,评价其肿瘤生长情况、转移情况,以期建立更稳定、更接近于人肺癌生长情况的小鼠肺癌原位模型。方法将处于对数生长期的Lewis肺癌细胞混悬于Matrigel中,接种于C57BL/6近交系小鼠左肺内。分别于第4、7、10、13、16天各处死5只小鼠,观察其局部成瘤率、肿瘤生长情况、中位生存期及肿瘤转移情况,并对各阶段小鼠行肺部,肝脏,肾脏,脾脏病理切片检查。结果术后第7天解剖的5只小鼠中,3只小鼠肺上可见小的瘤结节形成,其余2只肺上未见肉眼成瘤,行病理HE染色检查在显微镜下可见2只小鼠肺脏有小的瘤结节形成。术后第10天以后处死的所有小鼠肺上均有肉眼成瘤,术后第13天,所有小鼠肺原位成瘤并伴有血性胸腔积液、胸腔内转移。术后第25天,有1只小鼠出现上述转移的同时还出现了心包膜转移及肾脏远处转移。5只小鼠生存期分别为17 d、20 d、22 d、22 d、25 d,小鼠中位生存期为21.2 d（17~25 d）。成瘤率100%。结论利用Matrigel法成功建立小鼠Lewis肺癌原位模型,稳定性好,成瘤率高,并具有远处转移的特性,更接近于人肺癌的发生、发展过程。     

Impact of CD39 and purinergic signalling on the growth and metastasis of colorectal cancer

Despite improvements in prevention and management of colorectal cancer (CRC), uncontrolled tumor growth with metastatic spread to distant organs remains an important clinical concern. Genetic deletion of CD39, the dominant vascular and immune cell ectonucleotidase, has been shown to delay tumor growth and blunt angiogenesis in mouse models of melanoma, lung and colonic malignancy. Here, we tested the influence of CD39 on CRC tumor progression and metastasis by investigating orthotopic transplanted and metastatic cancer models in wild-type BALB/c, human CD39 transgenic and CD39 deficient mice. We also investigated CD39 and P2 receptor expression patterns in human CRC biopsies. Murine CD39 was expressed by endothelium, stromal and mononuclear cells infiltrating the experimental MC-26 tumors. In the primary CRC model, volumes of tumors in the subserosa of the colon and/or rectum did not differ amongst the treatment groups at day 10, albeit these tumors rarely metastasized to the liver. In the dissemination model, MC-26 cell line-derived hepatic metastases grew significantly faster in CD39 over-expressing transgenics, when compared to CD39 deficient mice. Murine P2Y2 was significantly elevated at both mRNA and protein levels, within the larger liver metastases obtained from CD39 transgenic mice where changes in P2X7 levels were also noted. In clinical samples, lower levels of CD39 mRNA in malignant CRC tissues appeared associated with longer duration of survival and could be linked to less invasive tumors. The modulatory effects of CD39 on tumor dissemination and differential levels of CD39, P2Y2 and P2X7 expression in tumors suggest involvement of purinergic signalling in these processes. Our studies also suggest potential roles for purinergic-based therapies in clinical CRC.     

The epidermal growth factor-seven transmembrane (EGF-TM7) receptor CD97 is required for neutrophil migration and host defense

The epidermal growth factor-seven transmembrane (EGF-TM7) family is a group of seven-span transmembrane receptors predominantly expressed by cells of the immune system. Family members CD97, EGF module-containing mucin-like receptor (EMR) 1, EMR2, EMR3, EMR4, and EGF-TM7-latrophilin-related protein are characterized by an extended extracellular region with a variable number of N-terminal EGF-like domains. EGF-TM7 receptors bind cellular ligands as demonstrated by the interaction of CD97 with decay accelerating factor (CD55) and dermatan sulfate. Investigating the effect of newly generated mAb on the migration of neutrophilic granulocytes, we here report for the first time in vivo data on the function of CD97. In dextran sulfate sodium-induced experimental colitis, we show that homing of adoptively transferred neutrophils to the colon was significantly delayed when cells were preincubated with CD97 mAb. The consequences of this defect in neutrophil migration for host defense are demonstrated in a murine model of Streptococcus pneumoniae-induced pneumonia. Mice treated with CD97 mAb to EGF domain 1 (1B2) and EGF domain 3 (1C5) displayed a reduced granulocytic inflammatory infiltrate at 20 h after inoculation. This was associated with a significantly enhanced outgrowth of bacteria in the lungs at 44 h and a strongly diminished survival. Together, these findings indicate an essential role for CD97 in the migration of neutrophils.