Human breast cancer cell metastasis to long bone and soft organs of nude mice : a quantitative assay

Bone is a common metastatic site in human breast cancer (HBC). Since bone metastasis occurs very rarely from current spontaneous or experimental metastasis models of HBC cells in nude mice, an arterial seeding model involving the direct injection of the cells into the left ventricle has been developed to better understand the mechanisms involved in this process. We present here a sensitive polymerase chain reaction (PCR) method to detect and quantitate bone and soft organ metastasis in nude mice which have been intracardially inoculated with Lac Z transduced HBC cells. Amplification of genomically incorporated Lac Z sequences in MDA-MB-231-BAG HBC cells enables us to specifically detect these cells in mouse organs and bones. We have also created a competitive template to use as an internal standard in the PCR reactions, allowing us to better quantitate levels of HBC metastasis. The results of this PCR detection method correlate well with cell culture detection from alternate long bones from the same mice, and are more sensitive than gross Lac Z staining with X-gal or routine histology. Comparable qualitative results were obtained with PCR and culture in a titration experiment in which mice were inoculated with increasing numbers of cells, but PCR is more quantifiable, less time consuming, and less expensive. This assay can be employed to study the molecular and cellular aspects of bone metastasis, and could easily be used in conjunction with RT-PCR-based analyses of gene products which may be involved with HBC metastasis.

Correlation between extent of osteolytic damage and metastatic burden of human breast cancer metastasis in nude mice : real-time PCR quantitation

Orthotopic or intracardiac injection of human breast cancer cell lines into immunocompromised mice allows study of the molecular basis of breast cancer metastasis. We have established a quantitative real-time PCR approach to analyze metastatic spread of human breast cancer cells inoculated into nude mice via these routes. We employed MDA-MB-231 human breast cancer cells genetically tagged with a bacterial β-galactosidase (Lac-Z) retroviral vector, enabling their detection by TaqMan® real-time PCR. PCR detection was linear, specific, more sensitive than conventional PCR, and could be used to directly quantitate metastatic burden in bone and soft organs. Attesting to the sensitivity and specificity of the PCR detection strategy, as few as several hundred metastatic MDA-MB-231 cells were detectable in 100 μm segments of paraffin-embedded lung tissue, and only in samples adjacent to sections that scored positive by histological detection. Moreover, the measured real-time PCR metastatic burden in the bone environment (mouse hind-limbs, n = 48) displayed a high correlation to the degree of osteolytic damage observed by high resolution X-ray analysis (r2 = 0.972). Such a direct linear relationship to tumor burden and bone damage substantiates the so-called 'vicious cycle' hypothesis in which metastatic tumor cells promote the release of factors from the bone which continue to stimulate the tumor cells. The technique provides a useful tool for molecular and cellular analysis of human breast cancer metastasis to bone and soft organs, can easily be extended to other cell/marker/organ systems, and should also find application in preclinical assessment of anti-metastatic modalities.

lacZ transduced human breast cancer xenografts as an in vivo model for the study of invasion and metastasis

A number of human cancer cell lines have been described as being invasive and metastatic in immune incompetent animals. However, it is difficult to assess metastatic spread of a subcutaneously injected or inoculated cell line, since an exact detection of all microfoci of human tumour cells in the animals by usual histological procedures would require extensive sectioning of the whole animal. To overcome this problem, we transduced human breast cancer cells with a replication-defective Moloney murine leukaemia retroviral vector (M-MuLV) containing both neo(R) (neomycin resistance) and lacZ genes. The resulting cell lines were selected for antibiotic (G418) resistance, and cell-sorted for lacZ expression. lacZ continued to be expressed in cultured cells for at least 20 passages without further G418 selection. The lacE gene codes for β-D-galactosidase, and cells expressing this gene stain blue with the chromogenic substrate X-gal. The lacZ-expressing cells retained the pre-transduction ability to traverse Matrigel in vitro, to form subcutaneous tumours in nude mice, and to grow invasively with the formation of metastases. X-gal staining showed high specificity, staining the tumour cells but not the surrounding mouse tissue on either whole tissue blocks or histological sections. The staining procedure was highly sensitive, allowing detection of microfoci of human cancer cells, and quantitative estimation of the metastatic capacity of the cells. These results indicate that lacZ transduction of human tumour cells is a powerful means of studying human cancer cell invasion and metastases in vivo.

ST7-mediated suppression of tumorigenicity of prostate cancer cells is characterized by remodeling of the extracellular matrix

Multiple lines of evidence have provided compelling evidence for the existence of a tumor suppressor gene (TSG) on chromosome 7q31.1. ST7 may be the target of this genetic instability but its designation as a TSG is controversial. In this study, we show that, functionally, ST7 behaves as a tumor suppressor in human cancer. ST7 suppressed growth of PC-3 prostate cancer cells inoculated subcutaneously into severe combined immunodeficient mice, and increased the latency of tumor detection from 13 days in control tumors to 23 days. Re-expression of ST7 was also associated with suppression of colony formation under anchorage-independent conditions in MDA-MB-231 breast cancer cells and ST7 mRNA expression was downregulated in 44% of primary breast cancers. Expression profiling of PC-3 cells revealed that ST7 predominantly induces changes in genes involved in re-modeling the extracellular matrix such as SPARC, IGFBP5 and several matrix metalloproteinases. These data indicate that ST7 may mediate tumor suppression through modification of the tumor microenvironment.

Epithelial mesenchymal transition traits in human breast cancer cell lines parallel the CD44HI/CD24lO/-stem cell phenotype in human breast cancer

We review here the recently emerging relationship between epithelial-mesenchymal transition (EMT) and breast cancer stem cells (BCSC), and provide analyses of published data on human breast cancer cell lines, supporting their utility as a model for the EMT/BCSC state. Genome-wide transcriptional profiling of these cell lines has confirmed the existence of a subgroup with mesenchymal tendencies and enhanced invasive properties ('Basal B'/Mesenchymal), distinct from subgroups with either predominantly luminal ('Luminal') or mixed basal/luminal ('Basal A') features (Neve et al. Cancer Cell, 2006). A literature-derived EMT gene signature has shown specific enrichment within the Basal B subgroup of cell lines, consistent with their over-expression of various EMT transcriptional drivers. Basal B cell lines are found to resemble BCSC, being CD44highCD24low. Moreover, gene products that distinguish Basal B from Basal A and Luminal cell lines (Basal B Discriminators) showed close concordance with those that define BCSC isolated from clinical material, as reported by Shipitsin et al. (Cancer Cell, 2007). CD24 mRNA levels varied across Basal B cell lines, correlating with other Basal B Discriminators. Many gene products correlating with CD24 status in Basal B cell lines were also differentially expressed in isolated BCSC. These findings confirm and extend the importance of the cellular product of the EMT with Basal B cell lines, and illustrate the value of analysing these cell lines for new leads that may improve breast cancer outcomes. Gene products specific to Basal B cell lines may serve as tools for the detection, quantification, and analysis of BCSC/EMT attributes.

Association of MMP-2 activation potential with metastatic progression in human breast cancer cell lines independent of MMP-2 production

Background: Expression of matrix metalloproteinase-2 (MMP-2), the 72-kd type IV collagenase/gelatinase, by cancer cells has been implicated in metastasis through cancer cell invasion of basement membranes mediated by degradation of collagen IV. However, the abundance of this latent proenzyme in normal tissues and fluids suggests that MMP-2 proenzyme utilization is limited by its physiological activation rather than expression alone. We previously reported activation of this proenzyme by normal and malignant fibroblastoid cells cultured on collagen I (vitrogen) gels. Purpose: Our purposes in this study were 1) to determine whether MMP-2 activation is restricted to the more invasive human breast cancer cell lines and 2) to localize the activating mechanism. Methods: Zymography was used to monitor MMP-2 activation through detection of latent MMP-2 (72 kd) and mature species of smaller molecular weight (59 or 62 kd). Human breast cancer cell lines cultured on plastic, vitrogen, and other matrices were thus screened for MMP- 2 activation. Collagen I-cultured cells were exposed to cycloheximide, a protein synthesis inhibitor, or to protease inhibitors to determine the nature of the MMP-2-activating mechanism. Triton X-114 (TX-114) detergent extracts from cells cultured on collagen I or plastic were incubated with latent MMP-2 and analyzed by zymography to localize the MMP-2 activator. Results: MMP-2 activation was only induced by collagen I culture in the more aggressive, highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines (Hs578T, MDA-MB-436, BT549, MDA-MB-231, MDA- MB-435, MCF-7(ADR)) and was independent of MMP-2 production. MMP-2 activation was detected in cells cultured on collagen I gels but not in those cultured on gelatin gels, Matrigel, or thin layers of collagen I or IV, gelatin, or fibronectin. Collagen-induced activation was specific for the enzyme species MMP-2, since MMP-9, the 92-kd type IV collagenase/gelatinase, was not activatable under similar conditions. MMP-2 activation was inhibited by cycloheximide and was sensitive to a metalloproteinase inhibitor but not to aspartyl, serine, or cysteinyl protease inhibitors. MMP-2 activation was detected in the hydrophobic, plasma membrane-enriched, TX-114 extracts from invasive collagen I-cultured cells. Conclusion: Collagen I-induced MMP-2 activation is restricted to highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines, is independent of MMP-2 production, and is associated with metastatic potential. Our findings are consistent with plasma membrane localization of the activator. Implications: The MMP-2 activation mechanism may represent a new target for diagnosis, prognosis, and treatment of human breast cancer.

Tunable “Nano-Shearing” : A physical mechanism to displace nonspecific cell adhesion during rare cell detection

We report a tunable alternating current electrohydrodynamic (ac-EHD) force which drives lateran fluid motion within a few nanometers of an electrode surface. Because the magnitude of this fluid shear force can be tuned externally (e.g., via the application of an ac electric field), it provides a new capability to physically displace weakly (nonspecifically) bound cellular analytes. To demonstrate the utility of the tunable nanoshearing phenomenon, we present data on purpose-built microfluidic devices that employ ac-EHD force to remove nonspecific adsorption of molecular and cellular species. Here, we show that an ac-EHD device containing asymmetric planar and microtip electrode pairs resulted in a 4-fold reduction in nonspecific adsorption of blood cells and also captured breast cancer cells in blood, with high efficiency (approximately 87%) and specificity. We therefore feel that this new capability of externally tuning and manipulating fluid flow could have wide applications as an innovative approach to enhance the specific capture of rare cells such as cancer cells in blood.

Alternating current electrohydrodynamics induced nanoshearing and fluid micromixing for specific capture of cancer cells

We report a new tuneable alternating current (ac) electrohydrodynamics (ac-EHD) force referred to as “nanoshearing” which involves fluid flow generated within a few nanometers of an electrode surface. This force can be externally tuned via manipulating the applied ac-EHD field strength. The ability to manipulate ac-EHD induced forces and concomitant fluid micromixing can enhance fluid transport within the capture domain of the channel (e.g., transport of analytes and hence increase target–sensor interactions). This also provides a new capability to preferentially select strongly bound analytes over onspecifically bound cells and molecules. To demonstrate the utility and versatility of nanoshearing phenomenon to specifically capture cancer cells, we present proof-of-concept data in lysed blood using two microfluidic devices containing a long array of asymmetric planar electrode pairs. Under the optimal experimental conditions, we achieved high capture efficiency (e.g., approximately 90%; %RSD=2, n=3) with a 10-fold reduction in nonspecific dsorption of non-target cells for the detection of whole cells expressing Human Epidermal Growth Factor Receptor 2 (HER2). We believe that our ac-EHD devices and the use of tuneable nanoshearing phenomenon may find relevance in a wide variety of biological and medical applications.

Clinicopathological relevance of BRAF mutations in human cancer

BRAF represents one of the most frequently mutated protein kinase genes in human tumours. The mutation is commonly tested in pathology practice. BRAF mutation is seen in melanoma, papillary thyroid carcinoma (including papillary thyroid carcinoma arising from ovarian teratoma), ovarian serous tumours, colorectal carcinoma, gliomas, hepatobiliary carcinomas and hairy cell leukaemia. In these cancers, various genetic aberrations of the BRAF proto-oncogene, such as different point mutations and chromosomal rearrangements, have been reported. The most common mutation, BRAF V600E, can be detected by DNA sequencing and immunohistochemistry on formalin fixed, paraffin embedded tumour tissue. Detection of BRAF V600E mutation has the potential for clinical use as a diagnostic and prognostic marker. In addition, a great deal of research effort has been spent in strategies inhibiting its activity. Indeed, recent clinical trials involving BRAF selective inhibitors exhibited promising response rates in metastatic melanoma patients. Clinical trials are underway for other cancers. However, cutaneous side effects of treatment have been reported and therapeutic response to cancer is short-lived due to the emergence of several resistance mechanisms. In this review, we give an update on the clinical pathological relevance of BRAF mutation in cancer. It is hoped that the review will enhance the direction of future research and assist in more effective use of the knowledge of BRAF mutation in clinical practice.

PSMA-targeting iron oxide magnetic nanoparticles enhance MRI of preclinical prostate cancer

Aim Evaluate potential of newly-developed, biocompatible iron oxide magnetic nanoparticles (MNPs) conjugated with J591, an antibody to an extracellular epitope of prostate specific membrane antigen (PSMA), to enhance MRI of prostate cancer (PCa). Materials & Methods Specific binding to PSMA by J591-MNP was investigated in vitro. MRI studies were performed on orthotopic tumor-bearing NOD.SCID mice 2h and 24hr after intravenous injection of J591-MNPs, or non-targeting MNPs. Results and Conclusions In vitro, MNPs did not affect PCa cell viability, and conjugation to J591 did not compromise antibody specificity and enhanced cellular iron uptake. In vivo, PSMA-targeting MNPs increased MR contrast of tumors, but not by non-targeting MNPs. This provides proof-of-concept that PSMA-targeting MNPs have potential to enhance MR detection/localization of PCa.,

Enhancing MRI of prostate cancer using PSMA-targeting iron oxide magnetic nanoparticles

Introduction Novel imaging techniques for prostate cancer (PCa) are required to improve staging and real-time assessment of therapeutic response. We performed preclinical evaluation of newly-developed, biocompatible magnetic nanoparticles (MNPs) conjugated with J591, an antibody specific for prostate specific membrane antigen (PSMA), to enhance magnetic resonance imaging (MRI) of PCa. PSMA is expressed on ∼90% of PCa, including those that are castrate-resistant, rendering it as a rational target for PCa imaging. Materials and Methods The specificity of J591 for PSMA was confirmed by flow cytometric analysis of several PCa cell lines of known PSMA status. MNPs were prepared, engineered to the appropriate size, labeled with DiR fluorophore, and their toxicity to a panel of PC cells was assessed by in vitro Alamar Blue assay. Immunohistochemistry, fluorescence microscopy and Prussian Blue staining (iron uptake) were used to evaluate PSMA specificity of J591-MNP conjugates. In vivo MRI studies (16.4T MRI system) were performed using live immunodeficient mice bearing orthotopic LNCaP xenografts and injected intravenously with J591-MNPs or MNPs alone. Results MNPs were non-toxic to PCa cells. J591-MNP conjugates showed no compromise in specificity of binding to PSMA+ cells and showed enhanced iron uptake compared with MNPs alone. In vivo, tumour targeting (significant MR image contrast) was evident in mice injected with J591-MNPs, but not MNPs alone. Resected tumours from targeted mice had an accumulation of MNPs, not seen in normal control prostate. Conclusions Application of PSMA-targeting MNPs into conventional MRI has potential to enhance PCa detection and localization in real-time, improving patient management.

Tumor-suppressor gene promoter hypermethylation in saliva of head and neck cancer patients

Head and neck squamous cell carcinoma (HNSCC) accounts for a bulk of the oral and laryngeal cancers, the majority (70%) of which are associated with smoking and excessive drinking, major known risk factors for the development of HNSCC. In contrast to reports that suggest an inverse relationship between smoking and global DNA CpG methylation, hypermethylation of promoters of a number of genes was detected in saliva collected from patients with HNSCC. Using a sensitive methylation-specific polymerase chain reaction (MSP) assay to determine specific methylation events in the promoters of RASSF1A, DAPK1, and p16 genes, we demonstrate that we can detect tumor presence with an overall accuracy of 81% in the DNA isolated from saliva of patients with HNSCC (n = 143) when compared with the DNA isolated from the saliva of healthy nonsmoker controls (n = 31). The specificity for this MSP panel was 87% and the sensitivity was 80%(with a Fisher exact test P < .0001). In addition, the test panel performed extremely well in the detection of the early stages of HNSCCs, with a sensitivity of 94% and a specificity of 87%, and a high. concordance value of 0.8, indicating an excellent overall agreement between the presence of HNSCC and a positive MSP panel result. In conclusion, we demonstrate that the promoter methylation of RASSF1A, DAPK1, and p16 MSP panel is useful in detecting hypermethylation events in a noninvasive manner in patients with HNSCC.

miRNAs in head and neck cancer revisited

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cause of cancer mortality in the world and the 5th most commonly occurring cancer. Tobacco smoking, alcohol consumption and human papilloma virus (HPV) infections have been associated with the occurrence of HNSCC. Despite advances that have been made in HNSCC treatment, smoking-associated HNSCC patients still exhibit a poor 5 year survival rate (30-50 %) and a concomitant poor quality of life. The major clinical challenge to date lies in the early detection of dysplastic lesions,which can progress to malignancy. In addition, there are currently no tools available to monitor HNSCC patients for early stages of local recurrences or distant metastases. In the recent past, micro-RNAs (miRNA) have been assessed for their role in cancer initiation and progression, including HNSCC. It is now well-established that deregulation of these single stranded, small non-coding, 19-25 nt RNAs can e.g. enhance the expression of oncogenes or subdue the expression of tumor suppressor genes. The aims of this review are three-fold: first to retrieve from the literature miRNAs that have specifically been associated with HNSCC, second to group these miRNAs into those regulating tumor initiation, progression and metastasis, and third to discern miRNAs related to smoking-associated HNSCC versus HPV-associated HNSCC development. This review gives an overview on the miRNAs regulating the development of head and neck cancers. The ultimate establishment of miRNA expression profiles that are HNSCC specific, and miRNAs that orchestrate altered gene and protein expression levels in HNSCC, could pave the way for a better understanding of the mechanism underlying its pathogenesis and the development of novel, targeted therapies.

Delirium in advanced cancer : screening for the incidence on admission to an inpatient hospice unit

Background Delirium is a common underdiagnosed condition in advanced cancer leading to increased distress, morbidity, and mortality. Screening improves detection but there is no consensus as to the best screening tool to use with patients with advanced cancer. Objective To determine the incidence of delirium in patients with advanced cancer within 72 hours of admission to an acute inpatient hospice using clinical judgement and validated screening tools. Method One hundred consecutive patients with advanced cancer were invited to be screened for delirium within 72 hours of admission to an acute inpatient hospice unit. Two validated tools were used, the Delirium Rating Scale-Revised 98 (DRS-R-98) and the Confusion Assessment Method (CAM) shortened diagnostic algorithm. These results were compared with clinical assessment by review of medical charts. Results Of 100 consecutive admissions 51 participated and of these 22 (43.1%) screened positive for delirium with CAM and/or DRS-R-98 compared to 15 (29.4%) by clinical assessment. Eleven (21.6%) were identified as hypoactive delirium and 5 (9.8%) as subsyndromal delirium. Conclusion This study confirms that delirium is a common condition in patients with advanced cancer.While there remains a lack of consensus regarding the choice of delirium screening tool this study supports theCAMas being appropriate. Further research may determine the optimal screening tool for delirium enabling the development of best practice clinical guidelines for routinemedical practice.