Isolation and enumeration of circulating tumor cells (CTCs) are used to monitor metastatic disease progression and guide cancer therapy. However, currently available technologies are limited to cells expressing specific cell surface markers, such as epithelial cell adhesion molecule (EpCAM) or have limited specificity because they are based on cell size alone. We developed a device, ApoStream TM that overcomes these limitations by exploiting differences in the biophysical characteristics between cancer cells and normal, healthy blood cells to capture CTCs using dielectrophoretic technology in a microfluidic flow chamber. Further, the system overcomes throughput limitations by operating in continuous mode for efficient isolation and enrichment of CTCs from blood. The performance of the device was optimized using a design of experiment approach for key operating parameters such as frequency, voltage and flow rates, and buffer formulations. Cell spiking studies were conducted using SKOV3 or MDA-MB-231 cell lines that have a high and low expression level of EpCAM, respectively, to demonstrate linearity and precision of recovery independent of EpCAM receptor levels. The average recovery of SKOV3 and MDA-MB-231 cancer cells spiked into approximately 12 Â 10 6 peripheral blood mononuclear cells obtained from 7.5 ml normal human donor blood was 75.4% 6 3.1% (n ¼ 12) and 71.2% 6 1.6% (n ¼ 6), respectively. The intra-day and inter-day precision coefficients of variation of the device were both less than 3%. Linear regression analysis yielded a correlation coefficient (R

Purpose: NKG2D (natural killer group 2, member D) binds to cellular ligands of the MIC and ULBP/RAET family. These ligands have restricted expression in normal tissue, but are frequently expressed on primary tumors. The role of NKG2D ligands is thought to be important in carcinogenesis but its prognostic effect has not been investigated in such a large cohort. Experimental Design: In our study, 462 primary colorectal tumors were screened for the expression of all MIC/ULBP/RAET proteins and NK cell infiltration. Tumor microarray technology was used for the purpose of this investigation. Results: NKG2D ligands were expressed by the majority of colorectal tumors; however, the level of expression varied considerably. High expression of MIC (68 versus 56 months) or RAET1G (74 versus 62 months) showed improved patient survival. Tumors expressing high levels of MIC and RAET1G showed improved survival of 77 months over tumors that expressed high levels of one ligand or low levels of both. High-level expression of all ligands was frequent in tumor-node-metastasis stage I tumors, but became progressively less frequent in stages II, III, and IV tumors. Expression of MIC was correlated with NK cellular infiltration. Conclusion:The observations presented are consistent with an immunoediting mechanism that selects tumor cells that have lost or reduced their expression of NKG2D ligands. The combination of MIC and tumor-node-metastasis stage was found to be the strongest predictor of survival, splitting patients into eight groups and suggesting prognostic value in clinical assessment. Of particular interest were stage I patients with low expression of MIC who had a similar survival to stage III patients, and may be candidates for adjuvant therapy. (Clin Cancer Res 2009;15(22):6993-7002)

BackgroundCell surface NKG2D ligands (NKG2DL) bind to the activating NKG2D receptor present on NK cells and subsets of T cells, thus playing a role in initiating an immune response. We examined tumor expression and prognostic effect of NKG2DL in breast cancer patients.MethodsOur study population (n = 677) consisted of all breast cancer patients primarily treated with surgery in our center between 1985 and 1994. Formalin-fixed paraffin-embedded tumor tissue was immunohistochemically stained with antibodies directed against MIC-A/MIC-B (MIC-AB), ULBP-1, ULBP-2, ULBP-3, ULBP-4, and ULBP-5.ResultsNKG2DL were frequently expressed by tumors (MIC-AB, 50% of the cases; ULBP-1, 90%; ULBP-2, 99%; ULBP-3, 100%; ULBP-4, 26%; ULBP-5, 90%) and often showed co-expression: MIC-AB and ULBP-4 (p = 0.043), ULBP-1 and ULBP-5 (p = 0.006), ULBP-4 and ULBP-5 (p < 0.001). MIC-AB (p = 0.001) and ULBP-2 (p = 0.006) expression resulted in a statistically significant longer relapse free period (RFP). Combined expression of these ligands showed to be an independent prognostic parameter for RFP (p < 0.001, HR 0.41). Combined expression of all ligands showed no associations with clinical outcome.ConclusionsWe demonstrated for the first time that NKG2DL are frequently expressed and often co-expressed in breast cancer. Expression of MIC-AB and ULBP-2 resulted in a statistically significant beneficial outcome concerning RFP with high discriminative power. Combination of all NKG2DL showed no additive or interactive effect of ligands on each other, suggesting that similar and co-operative functioning of all NKG2DL can not be assumed. Our observations suggest that among driving forces in breast cancer outcome are immune activation on one site and tumor immune escape on the other site.

To date five ULBP/RAET (UL16-binding protein, also known as retinoic acid early transcript) genes, encoded on human chromosome 6q24.2-q25.3, have been shown to encode ligands of the activating immunoreceptor NKG2D. Here, we show that a sixth gene, ULBP6/RAET1L, is a polymorphic locus that expresses a functional transcript. ULBP6 had a more restricted expression profile in cell lines and primary human tissues than other NKG2D ligands, but expression was detected in several human papillomavirus-positive cervical carcinoma cell lines and was inducible on infection with human CMV. ULBP6 bound to recombinant NKG2D as well as the human CMV immune evasion molecule UL16. By confocal microscopy we show that UL16 retains ULBP6 inside the cell, preventing it from reaching the cell surface. Expression of ULBP6 on target cells induced a significant increase in NK-cell killing. Comparison of ULBP6 with ULBP4 and ULBP5 indicated that differences in recombinant NKG2D binding correlated with differences in NK-cell activation.Key words: Immune evasion . Infectious diseases . Innate immunity . NK cells . NK-cell ligandsSupporting Information available online IntroductionThe stimulatory immune receptor NKG2D (NK group 2, member D) plays an important role in anti-pathogen and anti-cancer immune responses [1]. In humans, NKG2D is expressed by all NK cells and almost all abT cells and gdT cells [1]. It is normally absent from CD4 1 T cells but can be induced in some circumstances, such as in rheumatoid arthritis patients [2] and on exposure to human CMV (HCMV) [3]. NKG2D signalling function is mediated exclusively through the adaptor molecule DAP10 in humans [4], whereas mouse NKG2D can associate with both DAP10 and DAP12 [5,6]. The downstream effects of NKG2D ligation are now recognised to depend on synergy with other cell surface receptors and cytokines, with IL-15 signalling recently being shown to have a particularly close association with the NKG2D-DAP10 complex [7,8].NKG2D ligands are a diverse array of cellular proteins, all of which have structural similarity to MHC class I molecules [9]. In humans NKG2D ligands are encoded in two gene families, MIC (MHC-class-I-polypeptide related sequence) and ULBP/RAET (UL16-binding protein, also known as retinoic acid early transcript) [10][11][12][13]. We will follow the ULBP nomenclature from this point. In mouse, NKG2D ligands comprise three members of the H60 family, five members of the Rae1 (retinoic acid early inducible transcript-1) family, and Mult-1 (murine ULBP-like transcript) [14][15][16][17].According to the initial models of NKG2D function, its ligands were thought to be largely absent from healthy cells, but their expression could be induced on viral infection and on tumourigenesis [1]. Ectopic expression of NKG2D ligand leads to anticancer immune responses to transplanted tumours in mice, and the NKG2D knockout mouse has an increased susceptibility to certain types of cancer [18][19][20]. The importance of NKG2D in antiviral immune responses is illustrated by the fact that ...

The human activating immune receptor, NKG2D, binds to a diverse array of cellular ligands of the MIC and unique long 16 (UL16)-binding protein (ULBP)/retinoic acid early transcript (RAET) family. NKG2D is thought to participate in anticancer immune responses. By using tissue microarrays representing over 300 patients with defined clinicopathological factors, we present the first comprehensive screen of the expression of all NKG2D ligands in primary ovarian cancers. NKG2D ligands were expressed by the majority of tumors; however, the level of expression varied considerably. By categorizing each tumor as having negative, low or high expression, it was shown that high expression of several NKG2D ligands is inversely correlated with disease survival. Patients whose tumors had high expression of RAET1E (p 5 0.037), ULBP1 (p 5 0.036) and ULBP3 (p 5 0.004) surviving a median of 11, 14 and 11 months, respectively, compared with disease-specific survival of 29, 30 and 25 months in patients whose tumors showed no expression of these ligands. These results contrast with previous findings showing that high level NKG2D ligand expression is associated with good prognosis in colorectal cancer and suggest a fundamental difference in the involvement of NKG2D-mediated immunity in these two types of cancer. By using multivariate analysis, the factors retaining independent prognostic significance were International Federation of Gynecologists and Obstetricians stage (p < 0.001), presence of residual disease (p 5 0.003), ULBP2 (p 5 0.042) and RAET1E (p 5 0.030).Ovarian cancer is the second most common gynecological malignancy and the fourth most common cause of cancerrelated death in women in England and Wales, 1 with less than 10% of cases of ovarian cancer occurring in women under the age of 45 years. The 5-year survival rate of 30% improved little between 1950 and 1999. However, improvements in diagnosis and treatment in recent years are predicted to bring the 5-year survival up to 38% for women diagnosed after 2001. 1 The clinical factors of patient age, tumor stage and residual disease after surgical debulking are standard predictors of prognosis but do not account for the variation seen between apparently similar tumors when using these variables.There is now increasing evidence that the immune system may have a role to play in controlling tumor development and growth. 2 In multivariate analysis on prognostic factors in ovarian cancer, several groups have shown that the presence or absence of intratumoral T cells [3][4][5] and, in particular, cytotoxic T cells 6,7 was an independent predictor of enhanced survival. In contrast, the presence of intratumor regulatory T cells was associated with a poor prognosis. 8,9 Studies with in vivo cancer models strongly suggest that the activating immune receptor NKG2D is involved in anticancer immune responses. [10][11][12][13][14] In humans, both primary tumors and tumor derived cell lines frequently express NKG2D ligands. [15][16][17] The C-type lectin-like receptor NKG2D is unique among...

The activity of cytotoxic lymphocytes is regulated by the opposing function of stimulatory and inhibitory cell surface receptors. According to the now classical model of Natural Killer (NK) cell activity, the ligands for inhibitory receptors are constitutively expressed on healthy cells but can be lost on infection and on malignant cells. Loss of inhibitory checks will then allow activating signals to predominate, forming the basis of 'missing self recognition'. Natural Killer Group 2D (NKG2D) is an important member of the cohort of activating receptors expressed on Natural Killer (NK) cells and subsets of T cells. Ligands for the NKG2D receptor comprise a diverse array of self-proteins structurally related to MHC class I molecules. Expression of NKG2D ligands can be induced in cells during infection with pathogens, tumourigenesis, and by stimuli such as DNA damage, oxidative stress, and heat shock. Consequently NKG2D has been widely described as participating in 'stressed self' or 'damaged self' recognition. However, a body of evidence has recently emerged to suggest that this intuitive model of NKG2D function may be an oversimplification. NKG2D ligand expression has now widely been reported on cells that could not be described as stressed or damaged. For example activated T cells can express NKG2D ligands, and constitutive expression of NKG2D ligands has been reported on normal myelomonocytic cells, dendritic cells, and epithelial cells of the gut mucosa. In this article we will review the literature suggesting that NKG2D may function to recognise non-stressed cells and discuss the role NKG2D ligands could be playing in apparently healthy cells.

BackgroundThe activating immunoreceptor NKG2D is expressed on Natural Killer (NK) cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised.Methodology/Principal FindingsWe pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy.Conclusions/SignificanceWe demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have very different cellular localisations that are likely to reflect unique functionality.

Infection with human cytomegalovirus (HCMV) modulates the expression of a number of cellular receptors and is known to inhibit expression of the epidermal growth factor receptor (EGFR), a cell surface receptor that can promote cell proliferation through a cascade of intracellular signalling events. We have examined the mechanisms by which HCMV mediates downregulation of EGFR expression and show that virus infection results in the profound upregulation of Wilms' Tumour 1 (WT1) protein, a transcription factor associated with the negative regulation of a number of growth factors and growth factor receptors, including EGFR. Moreover, chromatin immunoprecipitation experiments also show that HCMV infection results in increased binding of WT1 to the EGFR promoter. Finally, we show that depleting the cell of WT1 using small interfering RNA abrogates virus-mediated downregulation of EGFR. Taken together, our observations suggest that HCMV-mediated repression of EGFR expression results from a virusmediated increase in cellular WT1, a known pleiotropic regulator of mitogenesis, apoptosis and differentiation.