How Reliable Is Ki-67 Immunohistochemistry in Grade 2 Breast Carcinomas? A QA Study of the Swiss Working Group of Breast- and Gynecopathologists

Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist's assessment of tumor proliferation. The Swiss Working Group of Gyneco- and Breast Pathologists has surveyed inter- and intraobserver consistency of Ki-67-based proliferative fraction in breast carcinomas. Methods Five pathologists evaluated MIB-1-labeling index (LI) in ten breast carcinomas (G1, G2, G3) by counting and eyeballing. In the same way, 15 pathologists all over Switzerland then assessed MIB-1-LI on three G2 carcinomas, in self-selected or pre-defined areas of the tumors, comparing centrally immunostained slides with slides immunostained in the different laboratoires. To study intra-observer variability, the same tumors were re-examined 4 months later. Results The Kappa values for the first series of ten carcinomas of various degrees of differentiation showed good to very good agreement for MIB-1-LI (Kappa 0.56–0.72). However, we found very high inter-observer variabilities (Kappa 0.04–0.14) in the read-outs of the G2 carcinomas. It was not possible to explain the inconsistencies exclusively by any of the following factors: (i) pathologists' divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique, and (iv) the selection of the tumor area in which to count. Despite intensive confrontation of all participating pathologists with the problem, inter-observer agreement did not improve when the same slides were re-examined 4 months later (Kappa 0.01–0.04) and intra-observer agreement was likewise poor (Kappa 0.00–0.35). Conclusion Assessment of mid-range Ki-67-LI suffers from high inter- and intra-observer variability. Oncologists should be aware of this caveat when using Ki-67-LI as a basis for treatment decisions in moderately differentiated breast carcinomas

Microfluidics for understanding basic aspects of directed immune cell migration and translational research

Immune cell migration is essential for mounting protective immune responses, yet fundamental also to the pathogenesis of autoimmune diseases such as multiple sclerosis. Trafficking of immune cells is regulated by a complex interplay of chemokine receptors and chemokines. Advances in the field of microfluidics have expanded our possibilities to study chemotaxis – the directed migration towards soluble chemokines. Upon encounter of a danger signal, dendritic cells (DCs) maturate and upregulate among other processes the chemokine receptor CCR7 which enables homing of these cells to lymph nodes along gradients of immobilized CCL21. Chemotaxis of DCs has typically been linked to spatial sensing, with cells sensing concentration differences along their diameter. However, recent data suggest that DCs can also navigate by temporal sensing characterized by the detection of temporal changes of chemokine concentrations at specific subcellular sites. So far, no experimental systems have been available to ultimately test these two hypotheses. In this work, we aimed at assessing whether dendritic cells navigate by spatial or temporal sensing in gradients of soluble CCL19, the second ligand for CCR7. To this end, we established a microfluidic migration device which allowed us to expose dendritic cells to precisely controlled chemokine gradient scenarios and monitor cells by time-lapse microscopy including quantification of cell morphology. The gradient scenarios developed in this study were characterized by a sequence of reversing stable and dynamic chemokine gradients and the response to the second gradient was assessed. We observed that the majority of DCs migrating in a stable CCL19 gradient reoriented towards the opposite direction when the chemokine gradient was reversed. This was independent of the concentration of the second chemokine gradient and whether the second chemokine gradient was kept stable or was dynamically increased. These findings strongly suggest that dendritic cells have the capacity to navigate by spatial gradient sensing. Moreover, we observed that cell size as a simple, yet intriguing parameter is linked to the capacity to navigate in chemokine gradients. This finding that a measure of cell geometry is linked to cellular navigation is well compatible with the concept of spatial gradient sensing. In a second project we have established a workflow to assess the migration capacities of cells derived from clinical samples containing only a few hundred cells. This offers opportunities for translational research as for example the analysis of cell migration of cells derived from the cerebrospinal fluid of multiple sclerosis patients

Nano-scale microfluidics to study 3D chemotaxis at the single cell level

Directed migration of cells relies on their ability to sense directional guidance cues and to interact with pericellular structures in order to transduce contractile cytoskeletal- into mechanical forces. These biomechanical processes depend highly on microenvironmental factors such as exposure to 2D surfaces or 3D matrices. In vivo, the majority of cells are exposed to 3D environments. Data on 3D cell migration are mostly derived from intravital microscopy or collagen-based in vitro assays. Both approaches offer only limited controllability of experimental conditions. Here, we developed an automated microfluidic system that allows positioning of cells in 3D microenvironments containing highly controlled diffusion-based chemokine gradients. Tracking migration in such gradients was feasible in real time at the single cell level. Moreover, the setup allowed on-chip immunocytochemistry and thus linking of functional with phenotypical properties in individual cells. Spatially defined retrieval of cells from the device allows down-stream off-chip analysis. Using dendritic cells as a model, our setup specifically allowed us for the first time to quantitate key migration characteristics of cells exposed to identical gradients of the chemokine CCL19 yet placed on 2D vs in 3D environments. Migration properties between 2D and 3D migration were distinct. Morphological features of cells migrating in an in vitro 3D environment were similar to those of cells migrating in animal tissues, but different from cells migrating on a surface. Our system thus offers a highly controllable in vitro-mimic of a 3D environment that cells traffic in vivo.ISSN:1932-620

The Immune-Metabolic Basis of Effector Memory CD4+ T Cell Function under Hypoxic Conditions

Effector memory (EM) CD4(+) T cells recirculate between normoxic blood and hypoxic tissues to screen for cognate Ag. How mitochondria of these cells, shuttling between normoxia and hypoxia, maintain bioenergetic efficiency and stably uphold antiapoptotic features is unknown. In this study, we found that human EM CD4(+) T cells had greater spare respiratory capacity (SRC) than did naive counterparts, which was immediately accessed under hypoxia. Consequently, hypoxic EM cells maintained ATP levels, survived and migrated better than did hypoxic naive cells, and hypoxia did not impair their capacity to produce IFN-gamma. EM CD4(+) T cells also had more abundant cytosolic GAPDH and increased glycolytic reserve. In contrast to SRC, glycolytic reserve was not tapped under hypoxic conditions, and, under hypoxia, glucose metabolism contributed similarly to ATP production in naive and EM cells. However, both under normoxic and hypoxic conditions, glucose was critical for EM CD4(+) T cell survival. Mechanistically, in the absence of glycolysis, mitochondrial membrane potential (DeltaPsim) of EM cells declined and intrinsic apoptosis was triggered. Restoring pyruvate levels, the end product of glycolysis, preserved DeltaPsim and prevented apoptosis. Furthermore, reconstitution of reactive oxygen species (ROS), whose production depends on DeltaPsim, also rescued viability, whereas scavenging mitochondrial ROS exacerbated apoptosis. Rapid access of SRC in hypoxia, linked with built-in, oxygen-resistant glycolytic reserve that functionally insulates DeltaPsim and mitochondrial ROS production from oxygen tension changes, provides an immune-metabolic basis supporting survival, migration, and function of EM CD4(+) T cells in normoxic and hypoxic conditions

Quantification of microfluidic 2D and 3D migration.

<p>(A) Distribution of migration tracks of cells migrating on fibronectin (100 μg/mL) or in a collagen gel (1.7 mg/mL). Rose plot summarizing frequency of directions relative to the starting point. (B) Quantification of migration characteristics of cells migrating on fibronectin (100 μg/mL, FN) or in a collagen gel of various densities (col conc = collagen concentration). Chemotactic Index is a measure of chemotactic efficiency (calculated by dividing the distance from start to end point of cells in axis of the gradient by the total migration distance of every cell). Median and interquartile ranges shown, ρ values were calculated by Kruksal-Wallis test with Dunn’s multiple comparison test. Not significant (ns) ρ ≥ 0.05; * ρ < 0.05; **** ρ < 0.0001. (C) Representative images of DC morphology in microfluidic 2D and 3D environments and mouse ear tissue. Scale bar, 10 μm. (D) Quantification of cell morphology of cells migrating on fibronectin (100 μg/mL), in a collagen gel (1.7 mg/mL) or in mouse ear tissue. Elongation factor = cell length divided by its perpendicular cell width. FN = fibronectin, col = collagen 1.7 mg/mL, crawl-in = mouse ear crawl-in migration assay. Median and interquartile ranges shown, ρ values were calculated by Kruksal-Wallis test with Dunn’s multiple comparison test. ** ρ < 0.01; *** ρ < 0.001.</p

Reproducibility of microfluidic 3D migration assay.

<p>(A) Analysis of spontaneous migration of DCs in 4 distinct collagen preparations (collagen concentration: 1.7 mg/mL). Cells were tracked for 90 min during culture in cell culture medium only. Panel 1 exemplarily shows migration tracks plotted to a common starting point, migration characteristics are shown in panel 2–3. Chemotactic Index is a measure for chemotactic efficiency (calculated by dividing the distance from start to end point of cells in axis of the gradient by the total migration distance of every cell). (B) DCs were tracked for 90 min during diffusion-based soluble CCL19 gradient (maximal CCL19 concentrations: 5 μg/ml). Panel 1 exemplarily shows migration tracks plotted to a common starting point, migration characteristics are shown in panel 2/3. Median and interquartile ranges shown, ρ values were calculated by Kruksal-Wallis test with Dunn’s multiple comparison test. Not significant (ns) ρ ≥ 0.05.</p