Hydrogel microstructure live-cell array for multiplexed analyses of cancer stem cells, tumor heterogeneity and differential drug response at single-element resolution.

Specific phenotypic subpopulations of cancer stem cells (CSCs) are responsible for tumor development, production of heterogeneous differentiated tumor mass, metastasis, and resistance to therapies. The development of therapeutic approaches based on targeting rare CSCs has been limited partially due to the lack of appropriate experimental models and measurement approaches. The current study presents new tools and methodologies based on a hydrogel microstructure array (HMA) for identification and multiplex analyses of CSCs. Low-melt agarose integrated with type I collagen, a major component of the extracellular matrix (ECM), was used to form a solid hydrogel array with natural non-adhesive characteristics and high optical quality. The array contained thousands of individual pyramidal shaped, nanoliter-volume micro-chambers (MCs), allowing concomitant generation and measurement of large populations of free-floating CSC spheroids from single cells, each in an individual micro-chamber (MC). The optical live cell platform, based on an imaging plate patterned with HMA, was validated using CSC-enriched prostate and colon cancer cell lines. The HMA methodology and quantitative image analysis at single-element resolution clearly demonstrates several levels of tumor cell heterogeneity, including morphological and phenotypic variability, differences in proliferation capacity and in drug response. Moreover, the system facilitates real-time examination of single stem cell (SC) fate, as well as drug-induced alteration in expression of stemness markers. The technology may be applicable in personalized cancer treatment, including multiplex ex vivo analysis of heterogeneous patient-derived tumor specimens, precise detection and characterization of potentially dangerous cell phenotypes, and for representative evaluation of drug sensitivity of CSCs and other types of tumor cells.

Donut-shaped chambers for analysis of biochemical processes at the cellular and subcellular levels.

In order to study cell-cell variation with respect to enzymatic activity, individual live cell analysis should be complemented by measurement of single cell content in a biomimetic environment on a cellular scale arrangement. This is a challenging endeavor due to the small volume of a single cell, the low number of target molecules and cell motility. Micro-arrayed donut-shaped chambers (DSCs) of femtoliter (fL), picoliter (pL), and nanoliter (nL) volumes have been developed and produced for the analysis of biochemical reaction at the molecular, cellular and multicellular levels, respectively. DSCs are micro-arrayed, miniature vessels, in which each chamber acts as an individual isolated reaction compartment. Individual live cells can settle in the pL and nL DSCs, share the same space and be monitored under the microscope in a noninvasive, time-resolved manner. Following cell lysis and chamber sealing, invasive kinetic measurement based on cell content is achieved for the same individual cells. The fL chambers are used for the analysis of the same enzyme reaction at the molecular level. The various DSCs were used in this proof-of-principle work to analyze the reaction of intracellular esterase in both primary and cell line immune cell populations. These unique DSC arrays are easy to manufacture and offer an inexpensive and simple operating system for biochemical reaction measurement of numerous single cells used in various practical applications.

An information-theoretical model for breast cancer detection.

OBJECTIVES: Formal diagnostic modeling is an important line of modern biological and medical research. The construction of a formal diagnostic model consists of two stages: first, the estimation of correlation between model parameters and the disease under consideration; and second, the construction of a diagnostic decision rule using these correlation estimates. A serious drawback of current diagnostic models is the absence of a unified mathematical methodological approach to implementing these two stages. The absence of a unified approach makes the theoretical/biomedical substantiation of diagnostic rules difficult and reduces the efficacy of actual diagnostic model application. METHODS: The present study constructs a formal model for breast cancer detection. The diagnostic model is based on information theory. Normalized mutual information is chosen as the measure of relevance between parameters and the patterns studied. The "nearest neighbor" rule is utilized for diagnosis, while the distance between elements is the weighted Hamming distance. The model concomitantly employs cellular fluorescence polarization as the quantitative input parameter and cell receptor expression as qualitative parameters. RESULTS: Twenty-four healthy individuals and 34 patients (not including the subjects analyzed for the model construction) were tested by the model. Twenty-three healthy subjects and 34 patients were correctly diagnosed. CONCLUSIONS: The proposed diagnostic model is an open one, i.e. it can accommodate new additional parameters, which may increase its effectiveness.

Antineutrophil cytoplasmic autoantibodies penetrate into human polymorphonuclear leukocytes and modify their apoptosis.

OBJECTIVE: The interaction of extracellular anti-neutrophil cytoplasmic autoantibodies (ANCA) with neutrophilic granules may play an important role in the pathogenesis of ANCA-related disorders. It has been confirmed that apoptosis is an essential trigger associated with translocation of the cytoplasmic granules to the cell surface, and with the expression of ANCA antigens. Since cell penetration by autoantibodies and apoptosis may be associated processes, we tested the hypothesis that penetration of ANCA-autoantibodies into polymorphonuclear leukocytes (PMNs) has an effect on apoptosis and thereby can influence surface antigen expression. METHODS: PMNs were isolated from the blood of healthy volunteers and incubated in the presence of anti-proteinase3 (PR3) enriched IgG or normal human IgG. For each period of incubation (40 minutes or 12 hours) we evaluated: 1) PMN morphology by light microscopy (LM) and transmission electron microscopy (TEM) for general estimation of the apoptotic process, and 2) ANCA binding to the target antigen by immunogold electron microscopy (IgEM). RESULTS: Both normal and anti-PR3 IgG penetrate PMNs. The labeled PR3-ANCA were localized on PR3 granules, regardless of the granules' location within the cell, and in the sites where the PMN destruction processes were most expressed. The destructive processes showed extensive apoptotic characteristics, in contrast to PMNs penetrated by normal IgG. CONCLUSION: PR3 ANCA penetrate PMNs and, via the interaction between PR3-ANCA and PR3-containing granule components, initiate a modification of the apoptotic process.