癌症生物学

Cell signaling is highly integrated for the process of various cell activities. Although previous studies have shown how individual genes contribute to cell migration, it remains unclear how the integration of these signaling pathways is involved in the modulation of cell migration. In our two-hit migration screen, we revealed that serine-threonine kinase 40 (STK40) and mitogen-activated protein kinase (MAPK) worked synergistically, and the suppression of both genes could further lead to suppression in cell migration. Furthermore, based on our analysis of cellular focal adhesion (FA) parameters using MATLAB analysis, we are able to find out the synergistic reduction of STK40 and MAPK that further abolished the increased FA by shSTK40. While FA identification in previous studies includes image analysis using manual selection, our protocol provides a semi-automatic manual selection of FAs using MATLAB. Here, we provide a method that can shorten the amount of time required for manual identification of FAs and increase the precision for discerning individual FAs for various analyses, such as FA numbers, area, and mean signals.

Cell migration is an essential biological process for organisms, in processes including embryonic development, immune response, and cancer metastasis. To elucidate the regulatory machinery of this vital process, methods that mimic in vivo migration, including in vitro wound healing assay and random migration assay, are widely used for cell behavior investigation. However, several concerns are raised with traditional cell migration experiment analysis. First, a manually scratched wound often presents irregular edges, causing the speed analysis difficult. Second, only the migration speed of leading cells is considered in the wound healing assay. Here, we provide a reliable analysis method to trace each cell in the time-lapse images, eliminating the concern about wound shape and creating a more comprehensive understanding of cell migration—not only of collective migration speed but also single-cell directionality and coordination between cells.

Macropinocytosis is an evolutionarily conserved process, which is characterized by the formation of membrane ruffles and the uptake of extracellular fluid. We recently demonstrated a role for CYFIP-related Rac1 Interactor (CYRI) proteins in macropinocytosis. High-molecular weight dextran (70kDa or higher) has generally been used as a marker for macropinocytosis because it is too large to fit in smaller endocytic vesicles, such as those of clathrin or caveolin-mediated endocytosis. Through the use of an image-based dextran uptake assay, we showed that cells lacking CYRI proteins internalise less dextran compared to their wild-type counterparts. Here, we will describe a step-by-step experimentation procedure to detect internalised dextran in cultured cells, and an image pipeline to analyse the acquired images, using the open-access software ImageJ/Fiji. This protocol is detailed yet simple and easily adaptable to different treatment conditions, and the analysis can also be automated for improved processing speed.

The invasion of tumor cells into the neighboring blood vessels and lymph nodes is a vital step for distant metastasis. Traditionally, the invasive activity of growth factors (or the anti-invasive activity of drugs) is measured with the Boyden chamber assay. However, this assay has a few disadvantages like poor physiological relevance of transwell inserts and an inability to control chemokine gradients. The Boyden chamber assay is one of the most prevalent methods to measure the invasion of cancer cells. It would be advantageous to develop another assay that could validate the results of the Boyden chamber assay. With this in mind, our laboratory developed the spherical invasion assay (SIA) to measure the pro-invasive activity of human cancer cells. The SIA also circumvents some of the drawbacks of the Boyden chamber assay. The present manuscript measures the anti-invasive activity of the Src kinase inhibitor PP2 in A549 human non-small cell lung carcinoma (NSCLC) cells using the SIA. The SIA protocol is comprised of two steps. In the first step, A549 human NSCLC cells (treated or not with PP2) were mixed with Matrigel and seeded in the middle of an eight-well chamber slide. After 24 h, a second layer of Matrigel was overlaid over the first layer. Over the course of the next 24 h, the A549 cells invade from the primary to the secondary Matrigel layers. Subsequently, the cells are visualized by phase-contrast microscopy and the images obtained are quantified using ImageJ to calculate the anti-invasive activity of PP2 in A549 cells. The results of the SIA correlate well with Boyden chamber assays. The SIA may be adapted for multiple experimental designs, such as drug screening (to combat invasion and metastasis), measuring the pro-invasive activity of growth factors, and elucidating the signaling pathways underlying the pro-invasive/anti-invasive activity of biological modifiers.

Graphic abstract:

Diagrammatic illustration of the spherical invasion assay (Hurley et al., 2017). A. The first layer is comprised of human cancer cells mixed in a 1:1 suspension with Phenol Red containing Matrigel (represented as LAYER 1 in the figure). After 24 h, the cancer cells grow and extend up to the boundary of this first layer. B. A second layer of 1:1 solution Phenol Red-free Matrigel, in Phenol Red-free RPMI (represented as LAYER 2 in the figure) is added on top of the first Matrigel spot. The cells are incubated for 24 h at 37°C. C. Over these 24 h, the cancer cells invade from the primary layer into the secondary Matrigel layer. The chamber slides are observed by phase-contrast microscopy. D. A representative photograph of the images obtained by the SIA is shown. The black arrow indicates the cancer cells invading into the second layer of Matrigel. The dotted line represents the interface between the two layers. The distance to which the cells have traveled (into the secondary Matrigel layer) is measured at ten sites (for each photograph) in a randomized double-blind fashion by three independent observers, using NIH ImageJ Version 1.47. This process is repeated for three separate photographic fields per sample.

Cell migration is a vital process in the development of multicellular organisms. When deregulated, it is involved in many diseases such as inflammation and cancer metastisation. Some cancer cells could be stimulated using chemoattractant molecules, such as growth factor Heregulin β1. They respond to the attractant or repellent gradients through a process known as chemotaxis. Indeed, chemotactic cell motility is crucial in tumour cell dissemination and invasion of distant organs. Due to the complexity of this phenomenon, the majority of available in vitro methods to study the chemotactic motility process have limitations and are mainly based on endpoint assays, such as the Boyden chamber assay. Nevertheless, in vitro time-lapse microscopy represents an interesting opportunity to study cell motility in a chemoattracting gradient, since it generates large volume image-based information, allowing the analysis of cancer cell behaviours. Here, we describe a detailed time-lapse imaging protocol, designed for tracking T47D human breast cancer cell line motility, toward a gradient of Heregulin β1 in a Dunn chemotaxis chamber assay. The protocol described here is readily adapted to study the motility of any adherent cell line, under various conditions of chemoattractant gradients and of pharmacological drug treatments. Moreover, this protocol could be suitable to study changes in cell morphology, and in cell polarity.

Research on cell migration and interactions with the extracellular matrix (ECM) was mostly focused on 2D surfaces in the past. Many recent studies have highlighted differences in migratory behaviour of cells on 2D surfaces compared to complex cell migration modes in 3D environments. When embedded in 3D matrices, cells constantly sense the physicochemical, topological and mechanical properties of the ECM and adjust their behaviour accordingly. Changes in the stiffness of the ECM can have effects on cell morphology, differentiation and behaviour and cells can follow stiffness gradients in a process called durotaxis. Here we introduce a detailed protocol for the assembly of 3D matrices consisting of collagen I/fibronectin and embedding cells for live cell imaging. Further, we will show how the matrix can be stiffened via non-enzymatic glycation and how collagen staining with fluorescent dyes allows simultaneous imaging of both matrix and cells. This approach can be used to image cell migration in 3D microenvironments with varying stiffness, define cell-matrix interactions and the cellular response to changing ECM, and visualize matrix deformation by the cells.

Cell migration is a fundamental cellular process that plays a crucial role in many physioglogical and pathological processes such as wound healing or cancer metastasis. Many assays have been developed to examine cell migration, such as the wound healing or scratch assay, Boyden Chamber or transwell assay, and the method we will describe here, single cell migration assay. In this assay, cells are plated sparsely on a collagen coated plate and live cell imaging is performed over a period of 2 h at 1 frame per minute. After imaging is completed, cells are tracked manually using ImageJ by tracking movement of the centroid of the cell. These data points are then exported and overall distance travelled from frame to frame is determined and divided by total time imaged to determine speed of the cell. This method provides a quick way to examine effect of cellular manipulation on cell migration before proceeding to perform more complex assays.

The ability of cancer cells to migrate through a complex three-dimensional (3D) environment is a hallmark event of cancer metastasis. Therefore, an in vitro migration assay to evaluate cancer cell migration in a 3D setting is valuable to examine cancer progression. Here, we describe such a simple migration assay in a 3D collagen-fibronectin gel for observing cell morphology and comparing the migration abilities of cancer cells. We describe below how to prepare the collagen-fibronectin gel castings, how to set up time-lapse recording, how to draw single-cell trajectories from movies and extract key parameters that characterize cell motility, such as cell speed, directionality, mean square displacement, and directional persistence. In our set-up, cells are sandwiched in a single plane between two collagen-fibronectin gels. This trick facilitates the analysis of cell tracks, which are for the most part 2D, at least in the beginning, but in a 3D environment. This protocol has been previously published in Visweshwaran et al. (2018) and is described here in more detail.

Metastasis is a complex process that includes several steps: neoplastic progression, angiogenesis, cell migration and invasion, intravasation into nearby blood vessels, survival in the circulatory system, extravasation followed by homing into distant tissues, the formation of micrometastases, and finally the growth into macroscopic secondary tumors. This complexity makes metastases difficult to investigate and quantify in animal models. The chick embryo is a unique in vivo model that overcomes many limitations for studying the metastatic process, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located under the eggshell, that is receptive to the xenografting of mammalian tumor cells, including human. Since the chick embryo is naturally immunodeficient at this stage, the CAM can support the engraftment of tumor cells, and their growth therein can faithfully recapitulate most of the characteristics of the carcinogenic process including: growth, invasion, angiogenesis and colonization of distant tissues (Deryugina and Quigley, 2008; Zijlstra et al., 2002). The CAM sustains rapid tumor formation within 5-7 days after cancer cell grafting. This feature provides a unique experimental model for a rapid study of the intravasation and colonization steps of the metastatic cascade. Furthermore, using quantitative PCR to detect species-specific sequences, such as Alu, the chick embryo CAM model can be used to monitor and quantify the presence of the xenografted, ectopic tumor cells in distant tissues. Thus, the chick embryo model has proved a valuable tool for cancer research, in particular for the investigation of molecules and pathways involved in cancer metastasis and to analyze the response of metastatic cancer to potential therapies (Herrero et al., 2015; Casar et al., 2014). In this respect, the use of the rapid and quantitative spontaneous metastasis chick embryo model can provide an alternative approach to conventional mouse model systems for screening anti-cancer agents.

The high migration rate of tumor cells often results in poor prognosis for the survival of the patients. Here, we describe a protocol to measure the migration of cells using a quantitative assay. The relative tumor cell migration was measured using ThinCerts^TM cell culture inserts and a lactate dehydrogenase (LDH) assay to quantify the relative cell number. The quantification of the migration with the LDH kit is much more precise than other methods using i.e. crystal blue to count the cells.