Transitions from mono- to co- to tri-culture uniquely affect gene expression in breast cancer, stromal, and immune compartments.

Heterotypic interactions in cancer microenvironments play important roles in disease initiation, progression, and spread. Co-culture is the predominant approach used in dissecting paracrine interactions between tumor and stromal cells, but functional results from simple co-cultures frequently fail to correlate to in vivo conditions. Though complex heterotypic in vitro models have improved functional relevance, there is little systematic knowledge of how multi-culture parameters influence this recapitulation. We therefore have employed a more iterative approach to investigate the influence of increasing model complexity; increased heterotypic complexity specifically. Here we describe how the compartmentalized and microscale elements of our multi-culture device allowed us to obtain gene expression data from one cell type at a time in a heterotypic culture where cells communicated through paracrine interactions. With our device we generated a large dataset comprised of cell type specific gene-expression patterns for cultures of increasing complexity (three cell types in mono-, co-, or tri-culture) not readily accessible in other systems. Principal component analysis indicated that gene expression was changed in co-culture but was often more strongly altered in tri-culture as compared to mono-culture. Our analysis revealed that cell type identity and the complexity around it (mono-, co-, or tri-culture) influence gene regulation. We also observed evidence of complementary regulation between cell types in the same heterotypic culture. Here we demonstrate the utility of our platform in providing insight into how tumor and stromal cells respond to microenvironments of varying complexities highlighting the expanding importance of heterotypic cultures that go beyond conventional co-culture.

A negative selection methodology using a microfluidic platform for the isolation and enumeration of circulating tumor cells.

Circulating tumor cells (CTCs) exist in the peripheral blood stream of metastatic cancer patients at rates of approximately 1 CTC per billion background cells. In order to capture and analyze this rare cell population, various techniques exist that range from antibody-based surface marker positive selection to methods that use physical properties of CTCs to negatively exclude background cells from a CTC population. However, methods to capture cells for functional downstream analyses are limited due to inaccessibility of the captured sample or labeling techniques that may be prohibitive to cell function. Here, we present a negative selection method that leverages a Microfluidic Cell Concentrator (MCC) to allow collection and analysis of this rare cell population without needing cell adhesion or other labeling techniques to keep the cells within the chamber. Because the MCC is designed to allow collection and analysis of non-adherent cell populations, multiple staining steps can be applied in parallel to a given CTC population without losing any of the population. The ability of the MCC for patient sample processing of CTCs for enumeration was demonstrated with five patient samples, revealing an average of 0.31 CTCs/mL. The technique was compared to a previously published method - the ELISPOT - that showed similar CTC levels among the five patient samples tested. Because the MCC method does not use positive selection, the method can be applied across a variety of tumor types with no changes to the process.

Streamlining immunoassays with immiscible filtrations assisted by surface tension.

Immunoassays are utilized for a wide variety of clinical and biomedical research applications. In typical immunoassays, analytes are captured, labeled, and quantified on a single surface (e.g., the bottom of a well plate). In order to minimize the background, this type of assay must be washed multiple times between each of these steps to ensure residual reagents (e.g., unbound labeling antibody) are removed from the system. In this manuscript, the immunoassay is fundamentally reconfigured, such that each reagent is confined to its own well and no wash steps are required. Using immiscible filtration assisted by surface tension (IFAST), a technique developed for nucleic acid and whole cell purifications, immunoassays can be drastically simplified such that all reagent manipulation is performed at the start of the assay (i.e., no pipetting steps are necessary during the assay). Analytes are bound to paramagnetic particles via antibodies and drawn through oil barriers between four isolated compartments: (1) sample well, (2) primary antibody labeling well, (3) secondary antibody labeling well, and (4) readout buffer well. Using this technique, we have demonstrated repeatable detection of as little as 188 fg of protein. IFAST immunoassay functionality is demonstrated by detecting a well accepted prostate cancer biomarker, prostate specific antigen (PSA). Assay performance was assessed by measuring known concentrations of recombinant PSA protein. The assay was then used to measure PSA concentrations in conditioned media and human plasma samples.

Role of thioredoxin reductase 1 and thioredoxin interacting protein in prognosis of breast cancer.

INTRODUCTION: The purpose of this work was to study the prognostic influence in breast cancer of thioredoxin reductase 1 (TXNRD1) and thioredoxin interacting protein (TXNIP), key players in oxidative stress control that are currently evaluated as possible therapeutic targets. METHODS: Analysis of the association of TXNRD1 and TXNIP RNA expression with the metastasis-free interval (MFI) was performed in 788 patients with node-negative breast cancer, consisting of three individual cohorts (Mainz, Rotterdam and Transbig). Correlation with metagenes and conventional clinical parameters (age, pT stage, grading, hormone and ERBB2 status) was explored. MCF-7 cells with a doxycycline-inducible expression of an oncogenic ERBB2 were used to investigate the influence of ERBB2 on TXNRD1 and TXNIP transcription. RESULTS: TXNRD1 was associated with worse MFI in the combined cohort (hazard ratio = 1.955; P < 0.001) as well as in all three individual cohorts. In contrast, TXNIP was associated with better prognosis (hazard ratio = 0.642; P < 0.001) and similar results were obtained in all three subcohorts. Interestingly, patients with ERBB2-status-positive tumors expressed higher levels of TXNRD1. Induction of ERBB2 in MCF-7 cells caused not only an immediate increase in TXNRD1 but also a strong decrease in TXNIP. A subsequent upregulation of TXNIP as cells undergo senescence was accompanied by a strong increase in levels of reactive oxygen species. CONCLUSIONS: TXNRD1 and TXNIP are associated with prognosis in breast cancer, and ERBB2 seems to be one of the factors shifting balances of both factors of the redox control system in a prognostic unfavorable manner.

Micro-Raman detection of nuclear membrane lipid fluctuations in senescent epithelial breast cancer cells.

Originally identified in cultured cells, oncogenic cellular senescence is a growth-arrest mechanism which may inhibit tumor development by limiting the ability of cells to divide. However, literature shows that these cells secrete tumor-inducing and tumor-suppressing proteins leading to poor prognosis. Understanding the progression of oncogenic cellular senescence and associated mechanisms provides important implications for improving tumorigenesis therapeutic treatments. Micro-Raman spectroscopic imaging has grown in popularity as an imaging technique compared to the standard imaging predecessors and can be attributed to its numerous benefits such as no sample perturbation and the provision of direct chemical information. Through the use of label-free micro-Raman spectroscopy, control and senescent cells were noninvasively imaged. Resulting spectral images were processed using chemometric techniques, and average nuclei spectra from each sample set were compared. In turn, changes in the -cis and -trans unsaturated lipid isomer content were found to differ among proliferating and senescent cells. This may lead to increased nuclear fluidity and may contribute to the inability of senescent cells to complete the cell cycle. In the tumor environment, this detected increase in nuclear envelope fluidity could lead to downstream gene expression modifications and increased nucleo-cytoplasmic RNA translocation. Understanding nuclear envelope fluidity could provide insight into secretory profiles of senescent cells and their role in carcinogenesis, meriting further investigation into novel therapeutic technique development for oncogenic cellular senescence.

Identification of new reference genes for the normalisation of canine osteoarthritic joint tissue transcripts from microarray data.

BACKGROUND: Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RT-qPCR) is the most accurate measure of gene expression in biological systems. The comparison of different samples requires the transformation of data through a process called normalisation. Reference or housekeeping genes are candidate genes which are selected on the basis of constitutive expression across samples, and allow the quantification of changes in gene expression. At present, no reference gene has been identified for any organism which is universally optimal for use across different tissue types or disease situations. We used microarray data to identify new reference genes generated from total RNA isolated from normal and osteoarthritic canine articular tissues (bone, ligament, cartilage, synovium and fat). RT-qPCR assays were designed and applied to each different articular tissue. Reference gene expression stability and ranking was compared using three different mathematical algorithms. RESULTS: Twelve new potential reference genes were identified from microarray data. One gene (mitochondrial ribosomal protein S7 [MRPS7]) was stably expressed in all five of the articular tissues evaluated. One gene HIRA interacting protein 5 isoform 2 [HIRP5]) was stably expressed in four of the tissues evaluated. A commonly used reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was not stably expressed in any of the tissues evaluated. Most consistent agreement between rank ordering of reference genes was observed between Bestkeeper(c) and geNorm, although each method tended to agree on the identity of the most stably expressed genes and the least stably expressed genes for each tissue. New reference genes identified using microarray data normalised in a conventional manner were more stable than those identified by microarray data normalised by using a real-time RT-qPCR methodology. CONCLUSION: Microarray data normalised by a conventional manner can be filtered using a simple stepwise procedure to identify new reference genes, some of which will demonstrate good measures of stability. Mitochondrial ribosomal protein S7 is a new reference gene worthy of investigation in other canine tissues and diseases. Different methods of reference gene stability assessment will generally agree on the most and least stably expressed genes, when co-regulation is not present.

Expression profiling of select cytokines in canine osteoarthritis tissues.

The objective of this study was to investigate the level of expression of five cytokines in four different articular tissues from the joints of dogs with and without osteoarthritis (OA). Articular tissues were harvested from the stifle (fat, cranial cruciate ligament, synovial membrane) or hip (articular cartilage) from eight dogs with OA secondary to cranial cruciate ligament disease (stifle) or hip dysplasia (hip), undergoing routine surgical treatment for the condition, and from five dogs euthanatized without orthopaedic disease. The mRNA transcript numbers for interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and interleukin-17 (IL-17) were determined by quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR). Increased expression of IL-1beta, IL-6 and IL-10 in OA synovial membrane, increased expression of IL-1beta and IL-6 in ruptured (OA) ligament, and reduced expression of IL-8 in OA synovial membrane were identified. Cytokine expression was detected in multiple tissues within the articular joint, but differential expression in OA was detected primarily in the synovial membrane and cranial cruciate ligament.