Manfred Kubbies

Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis.

Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round-or conical-bottom plates in standard medium and centrifuged for 10 min at 1000g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.

Reexpression of the TJ protein CLDN1 induces apoptosis in breast tumor spheroids

Members of the claudin family together with occludin are the major constituents of the tight junction (TJ) complex. The human homologue of the murine CLDN1, previously called SEMP1, was identified by differential expression analysis, and the CLDN1 mRNA was found to be downregulated or completely lost in human breast cancer cells in vitro. Retroviral‐induced CLDN1 reexpression in breast cancer cells results in plasma membrane homing of the protein and reconstitution of paracellular flux inhibition, which is not dependent on the presence of occludin protein. In this report, we investigated the physiologic role of CLDN1 in CLDN1‐transduced MDA‐MB 361 breast tumor cells in adherent 2D and suspension 3D spheroid cell cultures. Retroviral‐transduced bulk cultures were FACS‐sorted to enrich for 100% CLDN1‐positive clonal derivatives with similar expression levels of CLDN1 mRNA and protein. There was no difference in proliferation and cell death characteristics in 2D adherent cell cultures of CLDN1‐positive compared to control CLDN1‐negative and mock‐transduced cell cultures. In contrast, the majority of the CLDN1‐transduced derivatives displayed a significant elevation of apoptosis that became evident as early as 2 days after 3D spheroid culture onset. This elevated apoptosis was independent of the volume of established spheroids. The cellular immunofluorescence analysis of CLDN1 protein expression in transduced bulk cultures revealed a CLDN1‐positive subfraction with a heterogeneous pattern of membrane and cytosolic immunostaining. In the clonal MDA‐MB 361 CLDN1‐positive cultures, we found that a more prominent cell membrane localization correlated with a pronounced increase of apoptosis in tumor spheroids. In parallel, inhibition of the paracellular flux rate was observed. These findings support a potential role of the TJ protein CLDN1 in restricting nutrient and growth factor supplies in breast cancer cells, and they indicate that the loss of the cell membrane localization of the TJ protein CLDN1 in carcinomas may be a crucial step during tumor progression.

SEMP1, a senescence-associated cDNA isolated from human mammary epithelial cells, is a member of an epithelial membrane protein superfamily

We have cloned a human cDNA, SEMP1 (senescence-associated epithelial membrane protein 1), using differential display (DD) of mRNA. We compared mRNA expression profiles between cultured normal senescent human mammary epithelial cells (HMECs) and proliferating, early passage HMECs. From the amino acid sequence of the open reading frame (ORF) of the cDNA, we infer that the protein belongs to a family of membrane-associated, epithelial cell-specific proteins. The translation product has 91% identity to a mouse protein, claudin-1, a tight junction (TJ)-associated protein. SEMP1 mRNA is expressed in human tissues, including adult and fetal liver, pancreas, placenta, adrenals, prostate and ovary but at low or undetectable levels in a number of human breast cancer cell lines. SEMP1 is a member of a superfamily of epithelial membrane proteins (EMPs), which may have multiple potential functions, including maintenance and regulation of cell polarity and permeability, perhaps through mechanisms involving tight junctions.

Expression and targeting of the tight junction protein CLDN1 in CLDN1-negative human breast tumor cells

Claudins and occludin constitute the major transmembrane proteins of tight junctions (TJs). We have previously identified the human homologue of the murine Cldn1, CLDN1 (SEMP1) that is expressed in normal, mammary gland‐derived epithelial cells but is absent in most human breast cancer cell lines. To investigate the potential functions of CLDN1 protein in tumor and normal epithelial cells, we developed an I‐NGFR retroviral vector and monoclonal anti‐CLDN1 antibody. In subconfluent and confluent breast cancer cells, MDA‐MB‐435 and MDA‐MB‐361, endogenous CLDN1 expression was not detected by an anti‐CLDN1 monoclonal antibody by Western blot analysis or quantitative RT‐PCR. When CLDN1‐negative breast cancer cell lines were transduced with a CLDN1 retrovirus the cells express CLDN1 mRNA constitutively as shown by quantitative RT‐PCR. Immunofluorescence analyses of the CLDN1 retroviral transduced breast tumor cells using monoclonal antibodies against CLDN1 reveals a subcellular distribution at cell–cell contact sites similar to the CLDN1 homing pattern in T47‐D cells, which express endogenous CLDN1. This cell–cell contact co‐localization of CLDN1 was evident in CLDN1‐transduced breast tumor cells which fail to express occludin protein (MDA‐MB‐361 and MDA‐MB‐435) and express relatively little ZO‐1 protein (MDA‐MB‐435), suggesting that other proteins may be responsible for targeting of CLDN1 to cell–cell contact sites. The re‐expression of CLDN1 decreases the paracellular flux of 3 and 40 kDa dextran despite the absence of occludin in the MDA‐MB‐361 tumor cells. Our findings indicate that in CLDN1‐negative breast tumor cells, the basal protein partner requirements for physiological homing of the CLDN1 protein are intact, and that CLDN1 gene transfer and protein expression itself might be sufficient to exert a TJ‐mediate gate function in metastatic tumor cells even in the absence of other TJ‐associated proteins, such as occludin. J. Cell. Physiol. 191: 60–68, 2002.

Established breast cancer stem cell markers do not correlate with in vivo tumorigenicity of tumor-initiating cells.

The tumor-initiating capacity of primary human breast cancer cells is maintained in vitro by culturing these cells as spheres/aggregates. Inoculation of small cell numbers derived from these non-adherent cultures leads to rapid xenograft tumor formation in mice. Accordingly, injection of more differentiated monolayer cells derived from spheres results in significantly decelerated tumor growth. For our study, two breast cancer cell lines were generated from primary tumors and cultured as mammospheres or as their adherent counterparts. We examined the in vivo tumorigenicity of these cells by injecting serial dilutions into immunodeficient mice. Inoculation of 106 cells per mouse led to rapid tumor formation, irrespective of cell line or culture conditions. However, after injection of only 103 cells, solely sphere cells were highly tumorigenic. In vitro, we investigated differentiation markers, established breast CSC markers and conducted mRNA profiling. Cytokeratin 5 and 18 were increased in both monolayer cell types, indicating a more differentiated phenotype. All cell lines were CD24−/CD44+ and did not express CD133, CD326 or E-cadherin. ALDH1 activity was not detectable in any cell line. A verapamil-sensitive Hoechst side population was present in sphere cells, but there was no correlation with tumorigenicity in vivo. mRNA profiling did not reveal upregulation of relevant transcription factors. In vitro cell cycle kinetics and in vivo tumor doubling times displayed no difference between sphere and monolayer cultures. Our data indicate that intrinsic genetic and functional markers investigated are not indicative of the in vivo tumori-genicity of putative breast tumor-initiating cells.

High-Resolution Cell Cycle Analysis: The Flow Cytometric Bromodeoxyuridine-Hoechst Quenching Technique

Cell cycle analysis of in vitro cell cultures is of relevance in basic and clinical research in various fields of immunology, cell biology and oncology. Historically cell proliferation has been studied using cell-counting techniques or radioactive thymidine labeling of S phase cells. With the advent of single cell analysis via flow cytometry interphase G1, S and G2M cells were discernible. In addition, immunocytochemical techniques have been introduced selectively to label cycling Gl/S/GsM phase cells (proliferation markers such as proliferating cell nuclear antigen (PCNA) monoclonal antibodies, mAbs) or proliferating, BrdU-labeled S phase cells (BrdU mAbs) [1]

Cell Activation: Indo-1 Ratiometric Analysis of Intracellular Ionized Calcium

Intracellular ionized calcium (Ca2+) regulates various metabolic processes and is involved in signal transduction and cell activation. The intracellular concentration of Ca2+ in resting cells (100–200 nM) is far below the concentration of the extracellular environment. Most of the intracellular calcium, however, is bound in its nonionized form in the endoplasmic reticulum, mitochondria, cytosol, and cell membrane. Influx of Ca2+ into cells occurs through the action of voltage gated channels after membrane depolarization or by the action of receptor-gated channels. The excess of Ca2+ is pumped out of the cells by the action of a membrane-bound Ca2+ ATPase (for review see [1, 2].