Multi-Institutional Evaluation of Pathologists' Assessment Compared to Immunoscore.

BACKGROUND The Immunoscore (IS) is a quantitative digital pathology assay that evaluates the immune response in cancer patients. This study reports on the reproducibility of pathologists' visual assessment of CD3+- and CD8+-stained colon tumors, compared to IS quantification. METHODS An international group of expert pathologists evaluated 540 images from 270 randomly selected colon cancer (CC) cases. Concordance between pathologists' T-score, corresponding hematoxylin-eosin (H&E) slides, and the digital IS was evaluated for two- and three-category IS. RESULTS Non-concordant T-scores were reported in more than 92% of cases. Disagreement between semi-quantitative visual assessment of T-score and the reference IS was observed in 91% and 96% of cases before and after training, respectively. Statistical analyses showed that the concordance index between pathologists and the digital IS was weak in two- and three-category IS, respectively. After training, 42% of cases had a change in T-score, but no improvement was observed with a Kappa of 0.465 and 0.374. For the 20% of patients around the cut points, no concordance was observed between pathologists and digital pathology analysis in both two- and three-category IS, before or after training (all Kappa < 0.12). CONCLUSIONS The standardized IS assay outperformed expert pathologists' T-score evaluation in the clinical setting. This study demonstrates that digital pathology, in particular digital IS, represents a novel generation of immune pathology tools for reproducible and quantitative assessment of tumor-infiltrated immune cell subtypes

Cell migration in anisotropic media

A wide range of basic cellular process depends on cell motility that is fundamental for all eukaryotes. The ability of cells to migrate, adhere, and change shape requires most of the time external signals, although few cells respond primarily to internal cues. One of the most interesting and important response to external stimuli is chemotaxis. Chemotaxis, the directional movement of cells according to a concentration gradient of chemicals, is implicated in physiologically relevant phenomena such as inflammatory response, homeostatic circulation, and development and several disorders including infectious and allergic diseases, wound healing, angiogenesis, atherosclerosis, and tumor metastasis. However, despite the ubiquity and importance of chemotaxis, it remains a difficult process to study in vitro. The work carried out in this thesis present a novel chemotaxis assay in 3-D collagen gels in a direct-viewing chamber. Chemotaxis studies require a way to deliver chemicals to cells in a controlled gradient because cells need to be able to sense an increase in concentration of chemokine to direct their motion. In this chemotaxis assay a chemoattractant concentration gradient in the collagen gel sample seeded with cells is generated by diffusion trough a porous membrane. The diffusion process is monitored by fluorescence microscopy of FITC labelled dextran. Cell motion under the action of the chemoattractant gradient is followed by time-lapse video microscopy. Cell tracking is performed off-line by image analysis and the results are expressed in terms of a chemotactic index and velocity. The assay has been tested by using human neutrophils as a model

Comparison between fibroblast wound healing and cell random migration assays in vitro

Cell migration plays a key role in many biological processes, including cancer growth and invasion, embryogenesis, angiogenesis, inflammatory response, and tissue repair. In this work, we compare two well-established experimental approaches for the investigation of cell motility in vitro: the cell random migration (CRM) and the wound healing (WH) assay. In the former, extensive tracking of individual live cells trajectories by time-lapse microscopy and elaborate data processing are used to calculate two intrinsic motility parameters of the cell population under investigation, i.e. the diffusion coefficient and the persistence time. In the WH assay, a scratch is made in a confluent cell monolayer and the closure time of the exposed area is taken as an easy-to-measure, empirical estimate of cell migration. To compare WH and CRM we applied the two assays to investigate the motility of skin fibroblasts isolated from wild type and transgenic mice (TgPED) overexpressing the protein PED/PEA-15, which is highly expressed in patients with type 2 diabetes. Our main result is that the cell motility parameters derived from CRM can be also estimated from a time-resolved analysis of the WH assay, thus showing that the latter is also amenable to a quantitative analysis for the characterization of cell migration. To our knowledge this is the first quantitative comparison of these two widely used techniques

Cell trajectories projected on the XY plane and referred to the same origin.

<p>A: Random motion in absence of any chemical stimulus. B: In the presence of an IL 8 concentration gradient (C₀ = 50 µg/ml) a preferential direction is qualitatively evident: the cells migrate towards the negative Y direction, i.e. in the direction of the membrane, towards the chemoattractant gradient.</p

Fraction of motile cells as a function of the time for neutrophils A) from donor A, B) from donor B.

<p>The vertical line indicates the moment when the IL-8 solution was added to the chemoattractant reservoir.</p

Chemotaxis index as a function of time for neutrophils A) from donor A, B) from donor B.

<p>The vertical line indicates the time when the IL-8 solution was added to the chemoattractant reservoir.</p

Figure 6

<p>A: Average cell velocity components and modulus as a function of time for neutrophils from donor A. C: Average cell velocity components and modulus as a function of time for neutrophils from donor B. The vertical line indicates the moment when the IL-8 solution was added to the chemoattractant reservoir.</p

Images acquired at 5 consecutive focus positions within the collagen gel have been projected on the XY plane.

<p>The circles indicate the position of cells at two different times. Only the cells enclosed in a black circle move, while the white ones do not significantly change their position over the entire experiment. A: Time = 0. B: Time = 110 minutes. The complete trajectories described by motile cells are shown. The arrow indicates the direction of the chemoattractant concentration gradient ∇C.</p

Chemotaxis chamber.

<p>A: In the exploded view rendering of the chamber all the components are individually visible. B: In the assembled rendering, the membrane, sandwiched between two aluminum frames, is housed in the chamber, separating the sample well and the chemoattractant reservoir. C: Typical collagen gel morphology in confocal microscopy (63×, oil). Image size is 50 microns.</p

Average cell velocity module V calculated over the entire Pre IL-8 and Post IL-8 periods for each of the 63 cells from donor A.

<p>The continuous line is the average value, the error bar corresponds to the standard deviation for each population. Data are statistically significant (p<0.0001). In the inset the same data are plotted as a numerical distribution.</p