A survey of electrokinetically-driven microfluidics for cancer cells manipulation.

Cancer is one of the leading causes of annual deaths worldwide, accounting for nearly 10 million deaths each year. Metastasis, the process by which cancer spreads across the patient's body, is the main cause of death in cancer patients. Because the rising trend observed in statistics of new cancer cases and cancer-related deaths does not allow for an optimistic viewpoint on the future-in relation to this terrible disease-the scientific community has sought methods to enable early detection of cancer and prevent the apparition of metastatic tumors. One such method is known as liquid biopsy, wherein a sample is taken from a bodily fluid and analyzed for the presence of CTCs or other cancer biomarkers (e.g., growth factors). With this objective, interest is growing by year in electrokinetically-driven microfluidics applied for the concentration, capture, filtration, transportation, and characterization of CTCs. Electrokinetic techniques-electrophoresis, dielectrophoresis, electrorotation, and electrothermal and EOF-have great potential for miniaturization and integration with electronic instrumentation for the development of point-of-care devices, which can become a tool for early cancer diagnostics and for the design of personalized therapeutics. In this contribution, we review the state of the art of electrokinetically-driven microfluidics for cancer cells manipulation.

A Novel Cell-Assisted Enhanced Chemiluminescence Strategy for Rapid and Label-Free Detection of Tumor Cells in Whole Blood.

In this study, an interesting phenomenon was found where cells (including tumor and normal cells) managed to significantly enhance chemiluminescence (CL) signals. The possible reaction mechanism may be that cells can be severely damaged by CL substrates, and the released contents, possibly proteins (such as cytochrome c), can remarkably magnify CL owing to the increased production of singlet oxygen. More importantly, based on the above phenomena, a novel cell-assisted enhanced CL strategy was proposed for the rapid and label-free detection of tumor cells. The complexes of aptamer sgc8c and streptavidin-modified magnetic beads were employed to recognize and isolate target tumor cells from whole blood. The enhanced CL intensity, which was triggered directly by the captured cells, was measured. The proposed strategy exhibited a good detection performance with a linear range from 200 to 10,000 cells/mL. The analysis can be finished in ∼30 min, and the limit of detection was down to 100 cells/mL. The recoveries and relative standard deviations were 97.81-102.71% and 3.46-12.71%, respectively. Moreover, the established method can successfully distinguish the leukemia patients from healthy people. Therefore, it provides a novel, rapid, and simple method for the determination of tumor cells, which can be used in further practice.

Culturing substrates influence the morphological, mechanical and biochemical features of lung adenocarcinoma cells cultured in 2D or 3D.

Alternative models such as three-dimensional (3D) cell cultures represent a distinct milestone towards capturing the realities of cancer biology in vitro and reduce animal experimentation in the preclinical stage of drug discovery. Significant work remains to be done to understand how substrates used in in vitro alternatives influence cancer cells phenotype and drug efficacy responses, so that to accurately link such models to specific in vivo disease scenarios. Our study describes how the morphological, mechanical and biochemical properties of adenocarcinoma (A549) cells change in response to a 3D environment and varying substrates. Confocal Laser Scanning (LSCM), He-Ion (HIM) and Atomic Force (AFM) microscopies, supported by ELISA and Western blotting, were used. These techniques enabled us to evaluate the shape, cytoskeletal organization, roughness, stiffness and biochemical signatures of cells grown within soft 3D matrices (PuraMatrix™ and Matrigel™), and to compare them to those of cells cultured on two-dimensional glass substrates. Cell cultures are also characterized for their biological response to docetaxel, a taxane-type drug used in Non-Small-Cell Lung Cancer (NSCLC) treatment. Our results offer an advanced biophysical insight into the properties and potential application of 3D cultures of A549 cells as in vitro alternatives in lung cancer research.

Bottom-up proteomic analysis of single HCT 116 colon carcinoma multicellular spheroids.

RATIONALE: Proteomic analysis of single multicellular spheroids has not been previously reported. As three-dimensional cell cultures are an increasingly popular model system for biological research, there is interest in obtaining proteomic profiles of these samples. We investigated the proteome of single HCT 116 multicellular spheroids using protocols optimized for small sample sizes. METHODS: Six biological replicates were analyzed via microscopy for size. Total protein content was assessed via the bicinchoninic acid assay (BCA assay). Five separate biological replicate spheroids were analyzed via mass spectrometry in technical duplicate. An ultra-performance liquid chromatography (UPLC) system coupled with an LTQ Orbitrap Velos was used for peptide separation, analysis, and identification. RESULTS: The average diameter of six replicate HCT 116 spheroids was 940 ± 30 µm and the average total protein amount was determined to be 39 ± 4 µg. At least 1300 protein groups were identified in each single LC/MS/MS run with 10% of the material from each single spheroid loaded. Database search results showed variation between spheroid protein group identifications. Pearson correlations show that the disparity in identifications is due to random variations in spectra and protocol. CONCLUSIONS: We detected more than 1350 protein groups in each replicate HCT 116 spheroid. While some variation was detected between replicates, differences in the number of protein groups identified were determined to be the result of random variations in mass spectra acquisition.

[Therapeutic efficacy of dendritic cells pulsed by autologous tumor cell lysate in combination with CIK cells on advanced renal cell carcinoma].

OBJECTIVE: To investigate the clinical efficacy of dendritic cells (DCs) sensitization by autologous tumor cell lysate in combination with cytokine-induced killer (CIK) cells on patients with advanced renal cell carcinoma and detect their immune functions and adverse effects. METHODS: The study analyzed retrospectively 82 patients with advanced renal cell carcinoma admitted in our department from January 2011 to December 2013. Peripheral blood mononuclear cells (PBMCs) were isolated from the above patients. Adherent cells were cultured to produce DCs. The DCs were pulsed with autologous tumor cell lysate (Ag) to produce Ag-DC. T lymphocytes were cultured to prepare CIK. The Ag-DC was co-cultured with CIK to produce Ag-DC-CIK vaccine, and then the phenotypes of the DCs and the secretion of IL-12 were evaluated. CIK cell proliferation was determined, too. The 41 patients received the immunotherapy of Ag-DC-CIK, and the other 41 patients received CIK cell therapy alone. After 2 cycles of treatment, the changes of T cell subtypes and cytokines released in the peripheral blood were evaluated. The therapeutic outcomes were evaluated with the help of imageology. The adverse effects were also observed. RESULTS: Compared with the DCs alone, DCs pulsed with autologous tumor cell lysates expressed significantly surface molecules CD11c, CD83, CD86 and HLA-DR, and the release of IL-12 significantly increased. CIK cell proliferation was significantly enhanced after co-cultured with the Ag-DC. Meanwhile, the percentages of CD3⁺CD8⁺ cells and CD3⁺CD56⁺ cells went up significantly compared with the controls. Clinical responses of the Ag-DC-CIK treatment group were much better than those of the control group. No severe adverse effects were seen in the two groups. CONCLUSION: DCs derived from PBMCs of advanced renal cell carcinoma patients harboring the autologous tumor cell antigens can differentiate into mature DCs, which facilitate the proliferation of CIK cells. Ag-DC-CIK vaccine improves the immune status of patients with advanced renal cell carcinoma.

Nanosecond ratio imaging of redox states in tumor cell spheroids using light sheet-based fluorescence microscopy.

A new concept of three-dimensional imaging of tumor cell spheroids by light sheet-based fluorescence microscopy and nanosecond ratio imaging is described. Due to its low light dose and alternative excitation by two laser wavelengths (391 and 470 nm), this method maintains cell viability and permits recording of real-time kinetics. A genetically encoded sensor permits measurement of the redox state of glutathione and visualization of the impact of oxygen radicals. The pharmaceutically relevant system is tested upon addition of an oxidizing agent (H2O2), as well as upon addition of the apoptosis-inducing agent staurosporine.

Elevated physiological levels of folic acid can increase in vitro growth and invasiveness of prostate cancer cells.

OBJECTIVES: • To investigate the effects of different folic acid concentrations on the growth and invasiveness of prostate cancer cell lines. • To determine if observed changes are correlated with changes in levels of the potential prostate cancer biomarker, sarcosine, a byproduct of folate metabolism. MATERIALS AND METHODS: • The prostate cancer cell lines PC-3, LNCaP and DU145 were cultured in media containing 4, 20 or 100 nm of folic acid and assayed for growth over 9 days by counting viable cells at 3-day intervals, or for invasion by passage through a Matrigel-coated transwell membrane. • Cells grown in the different folic acid media were collected and subjected to metabolomic analysis by gas chromatography and mass spectrometry to measure levels of intracellular sarcosine. RESULTS: • The results show that higher levels of folic acid can increase cell growth in PC-3 and LNCaP prostate cancer cell lines, and may also increase the invasive capacity of PC-3, LNCaP and DU145 cells. • We did not observe a correlation between increased invasion from higher folic acid concentrations and levels of sarcosine, but there were significant changes in other metabolites in cells grown in higher levels of folic acid. CONCLUSION: • These findings suggest that folic acid has an important and potentially negative role in prostate cancer progression.

Holographic tissue dynamics spectroscopy.

Tissue dynamics spectroscopy uses digital holography as a coherence gate to extract depth-resolved quasi-elastic dynamic light scattering from inside multicellular tumor spheroids. The temporal speckle contrast provides endogenous dynamical images of proliferating and hypoxic or necrotic tissues. Fluctuation spectroscopy similar to diffusing wave spectroscopy is performed on the dynamic speckle to generate tissue-response spectrograms that track time-resolved changes in intracellular motility in response to environmental perturbations. The spectrograms consist of several frequency bands that range from 0.005 to 5 Hz. The fluctuation spectral density and temporal autocorrelations show the signature of constrained anomalous diffusion, but with large fluctuation amplitudes caused by active processes far from equilibrium. Differences in the tissue-response spectrograms between the proliferating outer shell and the hypoxic inner core differentiate normal from starved conditions. The differential spectrograms provide an initial library of tissue-response signatures to environmental conditions of temperature, osmolarity, pH, and serum growth factors.

Mesenchymal stem cells from multiple myeloma patients display distinct genomic profile as compared with those from normal donors.

It is an open question whether in multiple myeloma (MM) bone marrow stromal cells contain genomic alterations, which may contribute to the pathogenesis of the disease. We conducted an array-based comparative genomic hybridization (array-CGH) analysis to compare the extent of unbalanced genomic alterations in mesenchymal stem cells from 21 myeloma patients (MM-MSCs) and 12 normal donors (ND-MSCs) after in vitro culture expansion. Whereas ND-MSCs were devoid of genomic imbalances, several non-recurrent chromosomal gains and losses (>1 Mb size) were detected in MM-MSCs. Using real-time reverse transcription PCR, we found correlative deregulated expression for five genes encoded in regions for which genomic imbalances were detected using array-CGH. In addition, only MM-MSCs showed a specific pattern of 'hot-spot' regions with discrete (<1 Mb) genomic alterations, some of which were confirmed using fluorescence in situ hybridization (FISH). Within MM-MSC samples, unsupervised cluster analysis did not correlate with particular clinicobiological features of MM patients. We also explored whether cytogenetic abnormalities present in myelomatous plasma cells (PCs) were shared by matching MSCs from the same patients using FISH. All MM-MSCs were cytogenetically normal for the tested genomic alterations. Therefore we cannot support a common progenitor for myeloma PCs and MSCs.

The isolation of novel mesenchymal stromal cell chemotactic factors from the conditioned medium of tumor cells.

Bone marrow-derived mesenchymal stromal cells (MSCs) localize to solid tumors. Defining the signaling mechanisms that regulate this process is important in understanding the role of MSCs in tumor growth. Using a combination of chromatography and electrospray tandem mass spectrometry we have identified novel soluble signaling molecules that induce MSC chemotaxis present in conditioned medium of the breast carcinoma cell line MDA-MB231. Previous work has employed survey strategies using ELISA assay to identify known chemokines that promote MSC chemotaxis. While these studies provide valuable insights into the intercellular signals that impact MSC behavior, many less well-described, but potentially important soluble signaling molecules could be overlooked using these methods. Through the less directed method of column chromatography we have identified novel candidate MSC chemotactic peptides. Two proteins, cyclophilin B and hepatoma-derived growth factor were then further characterized and shown to promote MSC chemotaxis.

Evaluation of anti-tumor effects of tumor cell lysate enriched by HSP-70 against fibrosarcoma tumor in BALB/c mice.

The cytosolic members of the heat shock protein 70 (HSP-70) family have been shown to elicit protective cell mediated immunity in animal tumor models. The aim of this study was to investigate the effect of the HSP-70 enriched lysate of heated tumor cells as vaccines in cancer immunotherapy in the mouse model for WEHI-164 fibrosarcoma. Three animal bearing tumor groups were investigated: test group; vaccinated with enriched HSP-70 tumor lysate, control group I; vaccinated with tumor lysate only and control group II; received PBS. The results indicated that vaccinated mice in the test group had resulted in a significant reduction in tumor size and longer survival. To find the mechanism of these results, we measured the splenocytes proliferation, tumor infiltrated lymphocytes and cytotoxic activity of the splenocytes. The results indicated a significant increase in the proliferation of mouse splenocytes, a significant increase in the CD8+ lymphocytes as well as significant increase in the cytotoxic activity of splenocytes against the target cells in the test group. In addition, we analyzed the shifting of Th1/Th2 in all the groups. The results indicated a significant increase in the IFN-gamma production in the test group. These findings provided a useful therapeutic model for development of approaches to cancer treatments.

Identification of uPAR-positive chemoresistant cells in small cell lung cancer.

BACKGROUND: The urokinase plasminogen activator (uPA) and its receptor (uPAR/CD87) are major regulators of extracellular matrix degradation and are involved in cell migration and invasion under physiological and pathological conditions. The uPA/uPAR system has been of great interest in cancer research because it is involved in the development of most invasive cancer phenotypes and is a strong predictor of poor patient survival. However, little is known about the role of uPA/uPAR in small cell lung cancer (SCLC), the most aggressive type of lung cancer. We therefore determined whether uPA and uPAR are involved in generation of drug resistant SCLC cell phenotype. METHODS AND FINDINGS: We screened six human SCLC cell lines for surface markers for putative stem and cancer cells. We used fluorescence-activated cell sorting (FACS), fluorescence microscopy and clonogenic assays to demonstrate uPAR expression in a subpopulation of cells derived from primary and metastatic SCLC cell lines. Cytotoxic assays were used to determine the sensitivity of uPAR-positive and uPAR-negative cells to chemotherapeutic agents. The uPAR-positive cells in all SCLC lines demonstrated multi-drug resistance, high clonogenic activity and co-expression of CD44 and MDR1, putative cancer stem cell markers. CONCLUSIONS: These data suggest that uPAR-positive cells may define a functionally important population of cancer cells in SCLC, which are resistant to traditional chemotherapies, and could serve as critical targets for more effective therapeutic interventions in SCLC.

Deficient expression of NCR in NK cells from acute myeloid leukemia: Evolution during leukemia treatment and impact of leukemia cells in NCRdull phenotype induction.

Natural killer (NK) cells play an important role in tumor-cell clearance, particularly against leukemia, as shown by killer cell inhibitory receptor (KIR)-mismatched allogeneic stem cell transplantation. Analysis of in vitro IL-2-expanded NK cells from patients with myelocytic/monocytic acute myeloid leukemia (AML-NK cells) has revealed poor cytolytic functions because of deficient expression of pivotal activation molecules-the natural cytotoxicity receptors (NCRs) NKp30, NKp44, and NKp46. To exclude the possibility that this observation was caused by the in vitro amplification of a small NCR(dull) population, we analyzed the AML-NK phenotype directly, without any in vitro expansion. We first confirmed that the NCR(dull) phenotype was not an in vitro artifact. Moreover, analysis of a large population of AML patients allowed us to demonstrate that phenotype was not restricted to a French-American-British (FAB) subtype and was not associated with a particular cytogenetic abnormality. Our longitudinal study of AML patients showed that the NCR(dull) phenotype was acquired during leukemia development because we observed its complete (for NKp46) or partial (for NKp30) reversibility in patients achieving complete remission (CR). Reversibility of the NCR(dull) phenotype after CR suggested that leukemia cells might be involved in NCR down-regulation. In agreement with this hypothesis, direct contact between leukemic blasts and NK cells (but not leukemia-cell supernatants) induced loss or decrease in NKp30 and NKp46 expression while impeding NKp44 induction by IL-2. We excluded the major implication of TGF-beta in NCR down-regulation. Although the clinical antitumor value of NK cells is clearly demonstrated in allogeneic stem cell transplantation, the role of NK cells in autologous transplantation is not proved. Interestingly, we observed a correlation between the NCR(dull) phenotype and poor survival in AML patients, suggesting that NK-deficient activation caused by NCR down-regulation could play a role in patient outcome. The prognostic value of NCR expression is discussed, and pathophysiologic implication of the NCR phenotype will be further investigated in a larger study.

Short term culture of breast cancer tissues to study the activity of the anticancer drug taxol in an intact tumor environment.

BACKGROUND: Sensitivity of breast tumors to anticancer drugs depends upon dynamic interactions between epithelial tumor cells and their microenvironment including stromal cells and extracellular matrix. To study drug-sensitivity within different compartments of an individual tumor ex vivo, culture models directly established from fresh tumor tissues are absolutely essential. METHODS: We prepared 0.2 mm thick tissue slices from freshly excised tumor samples and cultivated them individually in the presence or absence of taxol for 4 days. To visualize viability, cell death, and expression of surface molecules in different compartments of non-fixed primary breast cancer tissues we established a method based on confocal imaging using mitochondria- and DNA-selective dyes and fluorescent-conjugated antibodies. Proliferation and apoptosis was assessed by immunohistochemistry in sections from paraffin-embedded slices. Overall viability was also analyzed in homogenized tissue slices by a combined ATP/DNA quantification assay. RESULTS: We obtained a mean of 49 tissue slices from 22 breast cancer specimens allowing a wide range of experiments in each individual tumor. In our culture system, cells remained viable and proliferated for at least 4 days within their tissue environment. Viability of tissue slices decreased significantly in the presence of taxol in a dose-dependent manner. A three-color fluorescence viability assay enabled a rapid and authentic estimation of cell viability in the different tumor compartments within non-fixed tissue slices. CONCLUSION: We describe a tissue culture method combined with a novel read out system for both tissue cultivation and rapid assessment of drug efficacy together with the simultaneous identification of different cell types within non-fixed breast cancer tissues. This method has potential significance for studying tumor responses to anticancer drugs in the complex environment of a primary cancer tissue.

Host plasminogen activator inhibitor-1 promotes human skin carcinoma progression in a stage-dependent manner.

Angiogenesis and tumor expansion are associated with extracellular matrix remodeling and involve various proteases such as the plasminogen (Plg)/plasminogen activator (PA) system. Recently, several experimental data have implicated the plasminogen activator inhibitor-1 (PAI-1) in tumor angiogenesis in murine systems. However, little is known about PAI-1 functions in human skin carcinoma progression. By generating immunodeficient mice (in Rag-1-/- or nude background) deleted for PAI-1 gene (PAI-1-/-), we have evaluated the impact of host PAI-1 deficiency on the tumorigenicity of two malignant human skin keratinocyte cell lines HaCaT II-4 and HaCaT A5-RT3 forming low-grade and high-grade carcinomas, respectively. When using the surface transplantation model, angiogenesis and tumor invasion of these two cell lines are strongly reduced in PAI-1-deficient mice as compared to the wild-type control animals. After subcutaneous injection in PAI-1-/- mice, the tumor incidence is reduced for HaCaT II-4 cells, but not for those formed by HaCaT A5-RT3 cells. These data indicate that PAI-1 produced by host cells is an important contributor to earlier stages of human skin carcinoma progression. It exerts its tumor-promoting effect in a tumor stage-dependent manner, but PAI-1 deficiency is not sufficient to prevent neoplastic growth of aggressive tumors of the human skin.

Water suppression without signal loss in HR-MAS 1H NMR of cells and tissues.

In cell and tissue samples, water is normally three orders of magnitude more abundant than other metabolites. Thus, water suppression is required in the acquisition of NMR spectra to overcome the dynamic range problem and to recover metabolites that overlap with the broad baseline of the strong water resonance. However, the heterogeneous cellular environment often complicates water suppression and the strong coupling of water to membrane lipids interferes with the NMR detection of membrane associated lipid components. The widely used water suppression techniques including presaturation and double pulsed field gradient selective echo result in more than a 70% reduction in membrane associated lipid components in proton spectra of cells and tissues compared to proton spectra acquired in the absence of water suppression. A water suppression technique based on the combination of selective excitation pulses and pulsed field gradients is proposed to use in the acquisition of high resolution MAS NMR spectra of tissue specimens and cell samples. This pulse sequence methodology enables efficient water suppression for intact cells and tissue samples and eliminates signal loss from cellular metabolites.

[Tumor markers of breast cancer]. / Markery nowotworowe raka piersi.

The most frequent cancer amongst women is that of the breast. Tumor markers may be helpful in the early diagnosis of breast cancer and the initial assessment of the extent of disease, as well as in monitoring tumor growth or volume reduction, and a recurrence of cancer. They have also been used for monitoring the clinical course of chemotherapy and radiotherapy. In this paper we focus on the role of tumor markers such as CA 15-3, CEA, and TPS in breast cancer diagnostics, including cytokines and molecular markers of carcinogenesis. We also show the prognostic significance of markers tested in breast cancer biopsies.

Proteomic analysis of dunning prostate cancer cell lines with variable metastatic potential using SELDI-TOF.

BACKGROUND: Surface enhanced laser desorption and ionization-time-of-flight (SELDI-TOF) is an evolving proteomic technology for improving biomarker discovery that allows for rapid and sensitive analysis of complex protein mixtures generated from body fluids, cells, and/or tissues. SELDI--based profiling identifies unique, differentially expressed proteins relating to specific cancer-related disease states. We utilized SELDI-TOF following pre-processing with molecular separation and chemical fractionation of cell membrane extracts from three Dunning rat prostate cancer cell lines of varying metastatic potential to search novel proteins that are differentially expressed. METHODS: Dunning rat cell sublines of variable (%) metastatic potential; G (0%), AT-1 (20%), and Mat-Ly-Lu (100%) were cultured in two different laboratories. Cell lysis was performed in a homogenation buffer (320 mM sucrose/50 mM Tris/0.5 mM PSMF) using Dounce homogenation. After centrifugation, the membrane pellet was washed 2x and then solublized in 2% CHAPS/8 M urea. This sample was further processed using positive pressure molecular ultrafiltration at 30 kDa or precipitation with 50% ammonium sulfate. Next, each sample was applied to an IMAC3-Ni ProteinChip (Ciphergen Biosystems, Freemont, CA) and analyzed using Ciphergen's Protein Biology System with protein peak analysis software. RESULTS: SELDI-TOF analysis differentiated the three Dunning rat cell sublines based upon protein concentration normalized profiles between 5,000 and 20,000 Da. The preparations from the three cells lines showed clear differences when the extracts from the metastatic sublines (AT-1 and MLL) were compared to the benign subline (G) for proteins with molecular weights of 9 kDa (decrease), 12 kDa (significant decrease), 14 kDa (decrease), and 17 kDa (significant gain). After pre-processing extracts with ammonium sulfate and molecular ultrafiltration, the molecular profile changes from one subline to the next became more apparent. Our results were reproducible using multiple runs including from Dunning cells cultured in a separate laboratory, and using different lots of SELDI ProteinChips. CONCLUSIONS: The application of SELDI-TOF to a series of Dunning rat prostate cancer cell lines illustrated apparent changes in protein profiles among the three cell lines with known differences in metastatic biologic activity. SELDI-TOF identified four reproducible changes in protein expression in the AT1 and MLL metastatic cell sublines. Three of the expression changes were manifested as decreases, but one protein (17 kDa) was over-expressed in the AT1 and MLL cell lines. Emphasis will be placed on the isolation, purification, and characterization of the 17 kDa over-expressed protein and its potential role in PCa metastasis.

LCC15-MB cells are MDA-MB-435: a review of misidentified breast and prostate cell lines.

Current stocks of the LCC15-MB cell line, which we originally isolated from a human breast-bone metastasis, were found to be genetically matched to the MDA-MB-435 cell line from the Lombardi Cancer Center (MDA-MB-435-LCC) using comparative genomic hybridisation, DNA microsatellite analysis and chromosomal number. LCC15-MB stocks used for our previously published studies as well as the earliest available LCC15-MB cells also showed identity to MDA-MB-435-LCC cells. The original karyotype reported for LCC15-MB cells was considerably different to that of MDA-MB-435 cells, indicating that the original LCC15-MB cells were lost to contamination by MDA-MB-435-LCC cells. Chromosome number is the simplest test to distinguish original LCC15-MB cells (n approximately 75) from MDA-MB-435 (n approximately 52). Collectively, our results prove that LCC15-MB cells currently available are MDA-MB-435 cells and we suggest their re-designation as MDA-MB-435-LCC15 cells. We also review the known misclassification of breast and prostate cancer cell lines to date and have initiated a register maintained at http://www.svi.edu.au/cell_lines_registry.doc.