Cancer metastasis directly eradicated by targeted therapy with a modified Salmonella typhimurium

Cancer metastasis is the life‐threatening aspect of cancer and is usually resistant to standard treatment. We report here a targeted therapy strategy for cancer metastasis using a genetically‐modified strain of Salmonella typhimurium. The genetically‐modified strain of S. typhimurium is auxotrophic for the amino acids arginine and leucine. These mutations preclude growth in normal tissue but do not reduce bacterial virulence in cancer cells. The tumor‐targeting strain of S. typhimurium, termed A1‐R, and expressing green fluorescent protein (GFP), was administered to both axillary lymph and popliteal lymph node metastasis of human pancreatic cancer and fibrosarcoma, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. The bacteria were delivered via a lymphatic channel to target the lymph node metastases and systemically via the tail vein to target the lung metastasis. The cancer cells expressed red fluorescent protein (RFP) in the cytoplasm and GFP in the nucleus linked to histone H2B, enabling color‐coded real‐time imaging of the bacteria targeting the metastatic tumors. After 7–21 days of treatment, the metastases were eradicated without the need of chemotherapy or any other treatment. No adverse effects were observed. This new strategy demonstrates the clinical potential of targeting and curing cancer metastasis with engineered bacteria without the need of toxic chemotherapy. J. Cell. Biochem. 106: 992–998, 2009. © 2009 Wiley‐Liss, Inc.     

PRC2/EED-EZH2 Complex Is Up-Regulated in Breast Cancer Lymph Node Metastasis Compared to Primary Tumor and Correlates with Tumor Proliferation In Situ

Background

Lymph node metastasis is a key event in the progression of breast cancer. Therefore it is important to understand the underlying mechanisms which facilitate regional lymph node metastatic progression.

Methodology/Principal Findings

We performed gene expression profiling of purified tumor cells from human breast tumor and lymph node metastasis. By microarray network analysis, we found an increased expression of polycomb repression complex 2 (PRC2) core subunits EED and EZH2 in lymph node metastatic tumor cells over primary tumor cells which were validated through real-time PCR. Additionally, immunohistochemical (IHC) staining and quantitative image analysis of whole tissue sections showed a significant increase of EZH2 expressing tumor cells in lymph nodes over paired primary breast tumors, which strongly correlated with tumor cell proliferation in situ. We further explored the mechanisms of PRC2 gene up-regulation in metastatic tumor cells and found up-regulation of E2F genes, MYC targets and down-regulation of tumor suppressor gene E-cadherin targets in lymph node metastasis through GSEA analyses. Using IHC, the expression of potential EZH2 target, E-cadherin was examined in paired primary/lymph node samples and was found to be significantly decreased in lymph node metastases over paired primary tumors.

Conclusions/Significance

This study identified an over expression of the epigenetic silencing complex PRC2/EED-EZH2 in breast cancer lymph node metastasis as compared to primary tumor and its positive association with tumor cell proliferation in situ. Concurrently, PRC2 target protein E-cadherin was significant decreased in lymph node metastases, suggesting PRC2 promotes epithelial mesenchymal transition (EMT) in lymph node metastatic process through repression of E-cadherin. These results indicate that epigenetic regulation mediated by PRC2 proteins may provide additional advantage for the outgrowth of metastatic tumor cells in lymph nodes. This opens up epigenetic drug development possibilities for the treatment and prevention of lymph node metastasis in breast cancer.     

Visualization of GFP-expressing tumors and metastasis in vivo

We have developed mouse models of metastatic cancer with genetically fluorescent tumors that can be imaged in fresh tissue, in situ, as well as externally. To achieve this capability, we have transduced the green fluorescent protein (GFP) gene, cloned from the bioluminescent jellyfish Aequorea victoria, into a series of human and rodent cancer cell lines that were selected in vitro to stably express GFP in vivo after transplantation to metastatic rodent models. Techniques were also developed for transduction of tumors by GFP in vivo. With this fluorescent tool, we detected and visualized for the first time tumors and metastasis in fresh viable tissue or in situ in host organs down to the single-cell level. GFP tumors on the colon, prostate, breast, brain, liver, lymph nodes, lung, pancreas, bone, and other organs can also be visualized externally, transcutaneously by quantitative whole-body fluorescence optical imaging. Real-time tumor and metastatic growth and angiogenesis and inhibition by representative drugs can be imaged and quantified for rapid antitumor, antimetastatic, and antiangiogenesis drug screening. The GFP-transfected tumor cells enabled a fundamental advance in the visualization of tumor growth and metastasis in real time in vivo.     

IL-10 promotes resistance to apoptosis and metastatic potential in lung tumor cell lines

Treatment of non-small cell lung carcinoma (NSCLC) remains at a disappointingly low success rate. Not only is metastatic spread common in NSCLC, but therapeutic success decreases dramatically once metastases are present. Understanding factors which contribute to poor prognosis in NSCLC is critical for development of more successful therapeutic approaches. Interleukin-10 (IL-10) expression has been shown in several studies to correlate with a poorer prognosis in NSCLC; however, the mechanisms by which IL-10 affects lung tumor growth and metastases are unclear. The goal of this study was to evaluate the effects of tumor-derived IL-10 on the growth and metastasis of lung cancer cells in a murine model. Lewis lung carcinoma cells were stably transfected with the chicken ovalbumin gene (cOVA) as a model tumor antigen (LL43 tumor cells) and subsequently transfected with the murine IL-10 gene (LL43-10 tumor cells). Subcutaneous growth of the LL43 tumor cells was not affected by expression of IL-10. However, LL43-10 tumors had a fourfold increase in tumor microvessel density, as indicated by CD31 staining. Metastatic potential was also increased in IL-10-expressing lung tumor cells, leading to a greater number of tumor cells in lymph nodes draining the primary tumor site. Finally, exposure of Lewis lung tumor cells in vitro to exogenous IL-10 dramatically increased their resistance to UV-induced apoptosis. These results indicate that a primary effect of IL-10 on lung cancer cells may be to increase their metastatic potential by promoting angiogenesis and resistance to apoptosis.     

Model for mediastinal lymph node metastasis produced by orthotopic intrapulmonary implantation of lung cancer cells in mice

This study is designed to establish a pulmonary tumor model to investigate the biology and therapy of lung cancer in mice. Current methods for forming a solitary intrapulmonary nodule and subsequent metastasis to mediastinal lymph nodes are not well defined. Lewis lung carcinoma cell (LLC) suspensions were orthotopically introduced into the lung parenchyma of C57/BL6 mice via a limited skin incision without thoracotomy followed by direct puncture through the intercostal space. The implantation process was performed within approximately 50 sec per mouse, and the operative mortality was less than 5%. Single pulmonary nodules developed at the implanted site in 93% of animals and subsequent mediastinal lymph nodes metastasis were observed in all mice that were succeeded to form a lung nodule after intrapulmonary implantation. The size of tumor nodule and the weight of mediastinal lymph node increased in a time-dependent manner. The mean survival time of mice implanted successfully with LLC cells was 21 +/- 2 days (range; 19-24 days). Histopathological analysis revealed that no metastatic tumor was detectable in the mediastinal lymph nodes on day 11, but metastatic foci at mediastinal lymph nodes were clearly observed on days 17 and 21 after implantation. Other metastases in distant organs or lymph nodes were not observed at 21 days after the implantation. Comparative studies with intrapleural and intravenous injections of LLC cells suggest that the mediastinal lymph node metastasis by intrapulmonary implantation is due to the release of tumor cells from the primary nodule, and not due to extrapulmonary leakage of cells. An intravenous administration of CDDP on day 1 after tumor implantation tended to suppress the primary tumor nodule and significantly inhibited the lymph node metastasis. Thus, a solitary pulmonary tumor nodule model with lymph node metastasis approximates clinical lung cancer, and may provide a useful basis for lung cancer research.     

Significance of Lymph Node Metastasis in Cancer Dissemination of Head and Neck Cancer

Lymph node metastasis (LNM) in many solid cancers is a well-known prognostic factor; however, it has been debated whether regional LNM simply reflects tumor aggressiveness or is a source for further tumor dissemination. Similarly, the metastatic process in head and neck cancer (HNC) has not been fully evaluated. Thus, we aimed to investigate the relative significance of LNM in metastatic cascade of HNC using functional imaging of HNC patients and molecular imaging in in vivo models. First, we analyzed ¹⁸Fluorodeoxyglucose positron emission tomography (PET) parameters of 117 patients with oral cancer. The primary tumor and nodal PET parameters were measured separately, and survival analyses were conducted on the basis of clinical and PET variables to identify significant prognostic factors. In multivariate analyses, we found that only the metastatic node PET values were significant. Next, we compared the relative frequency of lung metastasis in primary ear tumors versus lymph node (LN) tumors, and we tested the rate of lung metastasis in another animal model, in which each animal had both primary and LN tumors that were expressing different colors. As a result, LN tumors showed higher frequencies of lung metastasis compared to orthotopic primary tumors. In color-matched comparisons, the relative contribution to lung metastasis was higher in LN tumors than in primary tumors, although both primary and LN tumors caused lung metastases. In summary, tumors growing in the LN microenvironment spread to systemic sites more commonly than primary tumors in HNC, suggesting that the adequate management of LNM can reduce further systemic metastasis.     

Development of a Clinically-Precise Mouse Model of Rectal Cancer

Currently-used rodent tumor models, including transgenic tumor models, or subcutaneously growing tumors in mice, do not sufficiently represent clinical cancer. We report here development of methods to obtain a highly clinically-accurate rectal cancer model. This model was established by intrarectal transplantation of mouse rectal cancer cells, stably expressing green fluorescent protein (GFP), followed by disrupting the epithelial cell layer of the rectal mucosa by instilling an acetic acid solution. Early-stage tumor was detected in the rectal mucosa by 6 days after transplantation. The tumor then became invasive into the submucosal tissue. The tumor incidence was 100% and mean volume (±SD) was 1232.4 ± 994.7 mm³ at 4 weeks after transplantation detected by fluorescence imaging. Spontaneous lymph node metastasis and lung metastasis were also found approximately 4 weeks after transplantation in over 90% of mice. This rectal tumor model precisely mimics the natural history of rectal cancer and can be used to study early tumor development, metastasis, and discovery and evaluation of novel therapeutics for this treatment-resistant disease.     

Tumor-targeting amino acid auxotrophic <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

We have developed an effective bacterial cancer therapy strategy by targeting viable tumor tissue using Salmonella typhimurium auxotrophs that we have generated which grow in viable as well as necrotic areas of tumors. However, the auxotrophy severely restricts growth of these bacteria in normal tissue. The S. typhimurium A1-R mutant, which is auxotrophic for leu-arg and has high anti-tumor virulence, was developed in our laboratory. In vitro, A1-R infects tumor cells and causes nuclear destruction. A1-R was initially used to treat metastatic human prostate and breast tumors that had been orthotopically implanted in nude mice. Forty percent of treated mice were cured completely and survived as long as non-tumor-bearing mice. A1-R administered i.v. to nude mice with primary osteosarcoma and lung metastasis was highly effective, especially against metastasis. A1-R was also targeted to both axillary lymph and popliteal lymph node metastasis of human pancreatic cancer and fibrosarcoma, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. The bacteria were delivered via a lymphatic channel to target the lymph node metastases and systemically via the tail vein to target the lung metastasis. The metastases were cured without the need of chemotherapy or any other treatment. A1-R was administered intratumorally to nude mice with an orthotopically transplanted human pancreatic tumor. The primary pancreatic cancer regressed without additional chemotherapy or any other treatment. A1-R was also effective against pancreatic cancer liver metastasis when administered intrasplenically to nude mice. The approach described here, where bacterial monotherapy effectively treats primary and metastatic tumors, is a significant improvement over previous bacterial tumor-therapy strategies that require combination with toxic chemotherapy. Three promoter clones engineered in S. enterica typhimurium were identified to have enhanced expression in bacteria growing in tumors relative to those growing in the spleen. The expression of therapeutics in Salmonella under the regulation of one or more promoters that are activated preferentially in tumors has the potential to improve the efficacy of Salmonella tumor therapy. Exploitation of the tumor-killing capability of Salmonella has great promise for a new paradigm of cancer therapy.     

MAGE-A3/4 and NY-ESO-1 antigens expression in metastatic esophageal squamous cell carcinoma

In the present study we analyzed immunohistochemical expression of MAGE-A 3/4 and NY-ESO-1 in 55 samples of esophageal squamous cell carcinomas (ESCC) and their respective lymph node metastases. To our knowledge this is the first study to assess and compare the expression of these antigens in ESCC lymph node metastases. Fifty (90.9%) primary ESCC were positive for MAGE-A 3/4 and 53 (96.6%) were positive for NY-ESO-1. MAGE-A 3/4 was expressed in all lymph node metastases and the intensity of expression was high in a majority of cases. NY-ESO-1 was negative in 2 (7.1%) lymph nodes metastases, while the reaction was predominantly moderate in the positive group. In primary tumors MAGE-A 3/4 showed a significantly higher intensity of expression compared to NY-ESO-1 (P=0.047), while in lymph node metastases the intensity of expression was not significantly different (P=0.387). Primary tumors with and without lymph node metastases showed no significant differences in MAGE-A 3/4 (P=0.672) and NY-ESO-1 (P=0.444) expression. Intensity of MAGE-A 3/4 (P=0.461) and NY-ESO-1 (P=0.414) expression in primary tumors was not significantly different compared to the expression in their respective lymph nodes metastases. Expression of MAGE-A 3/4 in primary tumors showed significant positive correlation with primary tumor expression of NY-ESO-1 (P=0.021) but no significant correlation with the expression of MAGE-A 3/4 in lymph node metastases (P=0.056). Expression of NY-ESO-1 in primary tumors showed significant positive correlation with the expression of NY-ESO-1 in lymph node metastases (P=0.001) and significant negative correlation with patients’ age (P<0.001). Expression of MAGE-A 3/4 and NY-ESO-1 in primary tumors and lymph node metastases showed no significant correlation with prognostic parameters such as tumor grade and TNM stage (P>0.05). We have shown different levels of MAGE-A 3/4 and NY-ESO-1 expression in almost all specimens of primary tumor and lymph node metastases, suggesting that ESCC may be possible target of immunotherapy and anti-tumor vaccination. High levels of expression in lymph node metastases indicate possible clinical benefit of postoperative vaccine with MAGE-A3 and NY-ESO-1 in advanced stage of disease.     

Transforming growth factor-beta 1 overproduction in prostate cancer: effects on growth in vivo and in vitro.

We found previously that transforming growth factor-beta 1 (TGF beta 1) mRNA levels are markedly elevated in rat prostate cancer (Dunning R3327 sublines) compared to levels in normal prostate. Our goal was to determine whether elevated expression of TGF beta 1 is biologically relevant to prostate cancer growth in vivo. We chose as our model the R3327-MATLyLu prostate cancer epithelial cell line, which produces metastatic anaplastic tumors when reinoculated in vivo. Our approach was to stably transfect MATLyLu cells with an expression vector that codes for latent TGF beta 1 and to isolate subclones of cells that over-expressed TGF beta 1 mRNA. We also isolated a subclone of MATLyLu cells transfected with a control vector lacking the TGF beta 1 cDNA insert. We then studied the growth of these cells in vivo and in vitro. Twenty days after sc inoculation of 10(6) cells in vivo, TGF beta 1-overproducing MATLyLu tumors were 50% larger, markedly less necrotic, and produced more extensive metastatic disease (lung metastases in 73% of all lobes and lymph node metastases in 88% of animals) compared to control MATLyLu tumors (lung metastases, 21%; lymph node metastases, 7%). Thus, TGF beta 1 produced in vivo is biologically active and can promote prostate cancer growth, viability, and aggressiveness, perhaps via effects on the host and/or on the tumor cells themselves. When followed in vitro, TGF beta 1-overproducing cells became growth inhibited, but this effect was transient as cells subsequently resumed proliferating. Growth inhibition was due to TGF beta, because it could be prevented by TGF beta-neutralizing antibody. Therefore, prostate cancer cells can activate and respond to secreted latent TGF beta 1, and although the cells are transiently inhibited in vitro, there is no net inhibition of growth. The ability of the cells to respond to endogenously produced TGF beta 1 suggests that TGF beta 1 overexpression enhances tumor growth in vivo at least in part via an effect of TGF beta 1 on the tumor cells themselves.     

Expression of the insulin-like growth factor-I receptor in primary breast cancer and lymph node metastases: correlations with estrogen receptors alpha and beta.

Numerous laboratory studies and some epidemiological data have suggested the involvement of the insulin-like growth factor-I receptor (IGF-IR) in breast cancer development and progression. However, data on IGF-IR expression in human tissues, including breast cancer sections, are limited and often inconsistent. We therefore examined by immunohistochemistry the expression of IGF-IR in primary tumors and breast cancer metastases to lymph nodes, and correlated IGF-IR positivity with estrogen receptor (ER) status and selected clinicopathological features. We found that 1) IGF-IR was expressed in primary tumors as well as in lymph node metastases, but the expression in primary tumors was more frequent (56 % vs. 44.4 %); 2) IGF-IR expression in primary tumors was associated with negative node status (p < 0.033); 3) in node-negative primary tumors, IGF-IR positively correlated with ERbeta (p < 0.008; r = 0.538), but not with ERalpha, tumor size or grade; 4) both IGF-IR-positive and IGF-IR-negative primary tumors were found to produce IGF-IR-positive as well as IGF-IR-negative metastases; 5) in metastases, IGF-IR expression did not associate with ERalpha, ERbeta or any of the studied pathobiological markers. The results suggest that IGF-IR could become a viable pharmaceutical target in breast cancer therapy, but such therapy should be based on IGF-IR assessment in primary tumor and metastasis in each potential patient.     

A specific vaccine effective against stage I and stage II malignant disease in guinea pigs effect of variations in preparations and storage

Summary Guinea pigs, each with an established, syngeneic dermal line 10 tumor and lymph node metastases, were immunized by intradermal injection of a mixture of irradiated line 10 cells and an emulsion containing heat-killed BCG. Immunization eradicated 7- or 10-day-old dermal tumors (about 10 or 12 mm in diameter, respectively) and prevented growth of microscopic lymph node metastases. Fourteen-day-old dermal tumors (about 15 mm in diameter) were not rejected by immunization.Guinea pigs with stage II disease (21-day-old dermal tumors and palpable metastases in the first draining lymph node) were treated by excision of the dermal tumor and the first draining lymph node, and by specific immunization. This treatment eliminated tumor cells remaining in the second draining lymph nodes. The surgical treatment alone was not curative, palpable metastases in the second draining lymph nodes progressed and the animals died (some with visible lung metastases).Emulsions containing killed BCG were good adjuvants even after prolonged storage at 4° C, but lost most of their adjuvant activity after autoclaving or freezing.     

Growth of a human carcinoma (HEp3) in nude mice: rapid and efficient metastasis

Our aim was to identify conditions which would permit the development of spontaneous metastasis of a human tumor in nude mice in a rapid and predictable manner and to explore ways to quantitate metastasis. Using a human squamous carcinoma--HEp3--we determined that invasiveness and metastasis were influenced by the host. HEp3 cells grew very rapidly and without a significant lag period in Balb/c and NIH(S)-II nude mice kept in aseptic conditions; a much longer lag period was observed in NIH-Swiss mice kept in conventional conditions. The HEp3 tumor displayed a highly invasive behavior in N-NIH(S)-II mice, in which it invaded the body wall, gaining access to the peritoneal cavity. Microinvasion was noted in all strains of mice inoculated with HEp3 cells. To prolong survival of the mice until metastases became evident, primary tumors were excised when they weighed 1-2 gm. N-NIH(S)-II and Balb/c nude mice, maintained in germ-free conditions, were most receptive to the development of metastases-lung metastases developed in 80% of these mice. Over 60% of all metastases were present within 4 weeks following the removal of the primary. Only 26% of tumor bearing NIH-Swiss developed lung metastases. Lung metastases were observed in some mice in the absence of local microinvasion, local tumor recurrence, and regardless of the presence of lymph node involvement. They were also noted in mice from which primary tumors were not excised. We compared three methods of lung metastasis detection: histology, detection of tumor cells in the cultures of lung minces, and the measurement of the levels of human urokinase-type plasminogen activator directly in the lysates of lungs. The detection of tumor cells in cultures of lung minces appeared to be the most sensitive of these methods and the determination of enzyme in lung lysates seemed to hold most promise for a quantitative approach. These data indicate that, the type of tumor, as well as the genetic background and the maintenance conditions of the host, have to be carefully selected to ensure the successful outcome of the particular tumor-host interaction being studied. Adherence to these guidelines allowed us, in the case of the HEp3 tumor, to develop a rapid, predictable, and efficient model in which to study factors affecting metastasis of this human tumor.     

Fluorescent-Antibody Targeting of Insulin-Like Growth Factor-1 Receptor Visualizes Metastatic Human Colon Cancer in Orthotopic Mouse Models

Fluorescent-antibody targeting of metastatic cancer has been demonstrated by our laboratory to enable tumor visualization and effective fluorescence-guided surgery. The goal of the present study was to determine whether insulin-like growth factor-1 receptor (IGF-1R) antibodies, conjugated with bright fluorophores, could enable visualization of metastatic colon cancer in orthotopic nude mouse models. IGF-1R antibody (clone 24–31) was conjugated with 550 nm, 650 nm or PEGylated 650 nm fluorophores. Subcutaneous, orthotopic, and liver metastasis models of colon cancer in nude mice were targeted with the fluorescent IGF-1R antibodies. Western blotting confirmed the expression of IGF-1R in HT-29 and HCT 116 human colon cancer cell lines, both expressing green fluorescent protein (GFP). Labeling with fluorophore-conjugated IGF-1R antibody demonstrated fluorescent foci on the membrane of colon cancer cells. Subcutaneously- and orthotopically-transplanted HT-29-GFP and HCT 116-GFP tumors brightly fluoresced at the longer wavelengths after intravenous administration of fluorescent IGF-1R antibodies. Orthotopically-transplanted HCT 116-GFP tumors were brightly labeled by fluorescent IGF-1R antibodies such that they could be imaged non-invasively at the longer wavelengths. In an experimental liver metastasis model, IGF-1R antibodies conjugated with PEGylated 650 nm fluorophores selectively highlighted the liver metastases, which could then be non-invasively imaged. The IGF-1R fluorescent-antibody labeled liver metastases were very bright compared to the normal liver and the fluorescent-antibody label co-located with green fluorescent protein (GFP) expression of the colon cancer cells. The present study thus demonstrates that fluorophore-conjugated IGF-1R antibodies selectively visualize metastatic colon cancer and have clinical potential for improved diagnosis and fluorescence-guided surgery.     

Protein signatures associated with tumor cell dissemination in head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide. Strong prognostic indicators that predict development of distant metastases are the presence and number of lymph node metastases in the neck, and extranodal spread. Recently, it was shown in several studies that also the presence of disseminated tumor cells (DTC) in the bone marrow predicts development of distant metastases. We have investigated whether protein signatures could be detected in primary HNSCC that distinguish tumors that disseminate into the bone marrow from those that do not. Therefore, DTC-positive and -negative primary HNSCC tumors were analyzed by 2D-DIGE. A signature consisting of 51 differential protein spots was identified upon stratification for bone marrow status, which allowed a correct classification of DTC-positive and DTC-negative HNSCC tumors in 95% of cases, using hierarchical clustering. The most prominent feature within this signature was the down-regulation of CK19 in DTC-positive tumors. Our data show that tumor cell dissemination to the bone marrow, the onset of hematogenic metastasis, can be deduced from the protein profile in the primary tumor. The highly significant down-regulation of CK19 supports a model of epithelial-mesenchymal transition for tumors that show a high proclivity for hematogenic dissemination.     

Distinct Ezrin Truncations Differentiate Metastases in Sentinel Lymph Nodes from Unaffected Lymph Node Tissues,from Primary Breast Tumors,and from Healthy Glandular Breast Tissues

BACKGROUND: Lymph node metastasis status is a prognostic factor for further lymph node involvement and for patient survival in breast cancer patients. Frozen section analysis of lymph nodes is a reliable method for detection of macro-metastases. However, this method is far less effective in detecting micro-metastases, requesting improved diagnostic procedures. METHODS: We investigated expression and truncation of ezrin in (i) sentinel lymph node metastases, (ii) unaffected axillary lymph nodes, (iii) primary breast tumors, and (iv) healthy glandular breast tissues using 2D gel electrophoresis, SDS-PAGE, and mass spectrometry in addition to Western blotting. RESULTS: Full-length ezrin (E1; amino acids 1–586) is present in all four investigated tissues. Two truncated ezrin forms, one missing about the first hundred amino acids (E2a) and the other lacking about 150 C-terminal amino acids (E2b) were detectable in primary tumor tissues and in sentinel lymph node metastases but not in glandular tissues. Strikingly, an ezrin truncation (E3) which consists approximately of amino acids 238–586 was found strongly expressed in all sentinel lymph node metastases. Moreover, an N-terminal ezrin fragment (E4) that consists approximately of amino acids 1–273 was identified in sentinel lymph node metastases as well. CONCLUSIONS: We show for the first time the existence of tissue-dependent specific ezrin truncations. The distinguished strong Western blot staining of ezrin E3 in sentinel lymph node metastases underlines its capability to substantiate the occurrence of lymph node (micro)metastases in breast cancer patients.