Cancer Biology

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.

Platelets and their activation status play an essential role in cancer metastasis. Therefore, the anti-metastatic potential of antiplatelet drugs has been investigated for many years. However, the initial screening of these antiplatelet drugs to determine which agents can inhibit the interactions of platelets and tumor cells is very limited due to reliance upon expensive, time-consuming, and low-throughput animal experiments for screening. In vitro models of the platelet–tumor cell interaction can be a useful tool to rapidly screen multiple antiplatelet drugs and compare their ability to disrupt platelet–tumor cell interactions, while also identifying optimal concentrations to move forward for in vivo validation. Hence, we adopted methods used in platelet activation research to isolate and label platelets before mixing them with tumor cells (MDA-MB-231-RFP cells) in vitro in a static co-culture model. Platelets were isolated from other blood components by centrifugation, followed by fluorescent labeling using the dye CMFDA (CellTracker^TM Green). Labeling platelets allows microscopic observation of the introduced platelets with tumor cells grown in cell culture dishes. These methods have facilitated the study of platelet–tumor cell interactions in tissue culture. Here, we provide details of the methods we have used for platelet isolation from humans and mice and their staining for further interaction with tumor cells by microscopy and plate reader–based quantification. Moreover, we show the utility of this assay by demonstrating decreased platelet–tumor cell interactions in the presence of the T-Prostanoid receptor (TPr) inhibitor ifetroban. The methods described here will aid in the rapid discovery of antiplatelet agents, which have potential as anti-metastatic agents as well.

Key features

• Analysis of platelet–tumor cell binding dynamics.

• In vitro methods developed for measuring platelet–tumor cell binding to enable rapid testing of antiplatelet and other compounds.

• Complementary analysis of platelet–tumor cell binding by imaging and fluorimetry-based readings.

• Representative results screening the effect of the antiplatelet drug, ifetroban, on platelet–tumor cell binding using the protocol.

• Validation results were presented with both a TPr agonist and ifetroban (antagonist).

Graphical overview

Representative overview of the process to isolate and label platelets, incubate platelets and tumor cells in the presence of antiplatelet agents, and image and/or quantify platelet–tumor cell interactions.

Here, we present an in vivo drug screening protocol using a zebrafish model of metastasis for the identification of anti-metastatic drugs. A tamoxifen-controllable Twist1a-ER^T2 transgenic zebrafish line was established to serve as a platform for the identification. By crossing Twist1a-ER^T2 with xmrk (a homolog of hyperactive form of the epidermal growth factor receptor) transgenic zebrafish, which develop hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish show spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen and tail regions in five days, through induction of epithelial to mesenchymal transition (EMT). This rapid and high-frequency induction of cell dissemination makes it possible to perform an in vivo drug screen for the identification of anti-metastatic drugs targeting metastatic dissemination of cancer cells. The protocol evaluates the suppressor effect of a test drug on metastasis in five days, by comparing the frequencies of the fish showing abdominal and distant dissemination patterns in the test drug–treated group with those in the vehicle-treated group. Our study previously identified that adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1), has a suppressor effect on cell dissemination in the model. Furthermore, we validated that a pharmacologic and genetic inhibition of HSD11β1 suppressed metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model. Taken together, this protocol opens new routes for the identification of anti-metastatic drugs.

Graphical overview

Timing

Day 0: Zebrafish spawning

Day 8: Primary tumor induction

Day 11: Chemical treatment

Day 11.5: Metastatic dissemination induction in the presence of a test chemical

Day 16: Data analysis

The extracellular matrix (ECM) is a non-cellular network of macromolecules, which provides cells and tissues with structural support and biomechanical feedback to regulate cellular function, tissue tension, and homeostasis. Even subtle changes to ECM abundance, architecture, and organization can affect downstream biological pathways, thereby influencing normal cell and tissue function and also driving disease conditions. For example, in cancer, the ECM is well known to provide both biophysical and biochemical cues that influence cancer initiation, progression, and metastasis, highlighting the need to better understand cell–ECM interactions in cancer and other ECM-enriched diseases. Initial cell-derived matrix (CDM) models were used as an in vitro system to mimic and assess the physiologically relevant three-dimensional (3D) cell–ECM interactions. Here, we describe an expansion to these initial CDM models generated by fibroblasts to assess the effect of genetic or pharmacological intervention on fibroblast-mediated matrix production and organization. Additionally, we highlight current methodologies to quantify changes in the ultrastructure and isotropy of the resulting ECM and also provide protocols for assessing cancer cell interaction with CDMs. Understanding the nature and influence of these complex and heterogeneous processes can offer insights into the biomechanical and biochemical mechanisms, which drive cancer development and metastasis, and how we can target them to improve cancer outcomes.

Mature B-cell lymphomas are highly dependent upon the protective lymphoid organ microenvironment for their growth and survival. Targeting integrin-mediated homing and retention of the malignant B cells in the lymphoid organs, using the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, is a highly efficacious FDA-approved therapy for chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström macroglobulinemia (WM). Unfortunately, a significant subset of patients is intrinsically resistant to ibrutinib or will develop resistance upon prolonged treatment. Here, we describe an unbiased functional genomic CRISPR-Cas9 screening method to identify novel proteins involved in B-cell receptor–controlled integrin-mediated adhesion, which provides novel therapeutic targets to overcome ibrutinib resistance. This screening method is highly flexible and can be easily adapted to identify cell adhesion–regulatory proteins and signaling pathways for other stimuli, adhesion molecules, and cell types.

Graphical abstract:

Few models exist that allow for rapid and effective screening of anti-metastasis drugs. Here, we present a drug screening protocol utilizing gastrulation of zebrafish embryos for identification of anti-metastasis drugs. Based on the evidence that metastasis proceeds through utilizing the molecular mechanisms of gastrulation, we hypothesized that chemicals interrupting zebrafish gastrulation might suppress the metastasis of cancer cells. Thus, we developed a phenotype-based chemical screen that uses epiboly, the first morphogenetic movement in gastrulation, as a marker. The screen only needs zebrafish embryos and enables hundreds of chemicals to be tested in five hours by observing the epiboly progression of chemical-treated embryos. In the screen, embryos at the two-cell stage are firstly corrected and then developed to the sphere stage. The embryos are treated with a test chemical and incubated in the presence of the chemical until vehicle-treated embryos develop to the 90% epiboly stage. Finally, positive ‘hit’ chemicals that interrupt epiboly progression are selected by comparing epiboly progression of the chemical-treated and vehicle-treated embryos under a stereoscopic microscope. A previous study subjected 1,280 FDA-approved drugs to the screen and identified adrenosterone and pizotifen as epiboly-interrupting drugs. These were validated to suppress metastasis of breast cancer cells in mice models of metastasis. Furthermore, 11β-hydroxysteroid dehydrogenase 1 (HSD11β1) and serotonin receptor 2C (HTR2C), the primary targets of adrenosterone and pizotifen, respectively, promoted metastasis through induction of epithelial-mesenchymal transition (EMT). Therefore, this screen could be converted into a chemical genetic screening platform for identification of metastasis-promoting genes.

Graphical abstract:

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.