A Method to Study Migration and Invasion of Mouse Intestinal Organoids

Abstract

Colorectal cancer (CRC) is the third most common cancer worldwide and it is the second leading cause of cancer death. In CRC, as in most cancers, the formation of metastasis through the migration and invasion of cancer cells to distant organs is associated with a dismal prognosis. The study of the mechanisms associated with cancer, and, in particular, CRC, changed in the last decade due to the introduction of organoids. These represent a step forward in terms of complexity from cell lines and allowed the use of mouse models in cancer research to be limited. Although organoids faithfully model the cellular complexity of CRC, current protocols do not allow for the use of organoids in some crucial processes of metastasis, such as migration and invasion. In this study, a method to study migration and invasion using mouse intestinal organoids in vitro is presented. This protocol provides researchers with the opportunity to investigate the migratory behavior of organoid lines and study the impact of distinct mutations on the migratory and invasive capacity of cancer cells.

1. Introduction

CRC is one of the leading causes of cancer-related mortality. In 2020, two million new cases of CRC were diagnosed, ranking it as the third most common cancer worldwide [1]. It is well known that the development of CRC is a multistep process starting from an adenoma that progresses to carcinoma and eventually to a widespread metastatic disease [2]. In order to form metastasis, cancer cells need to invade the surrounding tissue and migrate to distant organs, such as the liver and lungs [3].

Although therapeutic strategies have improved in recent years, increasing the survival rate of CRC patients, the development of metastases represents a major negative predictive factor [3]. Therefore, there is an urgent need to better understand the biological processes that orchestrate metastasis in CRC.

To gain insight into metastatic CRC, various preclinical models have been developed. Mouse models are a valuable tool in cancer research, and many of them have been generated to study the different steps of CRC. Some of these CRC mouse models are able to form metastasis [4]. A good example is the KPN mouse model, which carries a tamoxifen inducible Cre recombinase under the control of the villin promoter, which is specific for the epithelial compartment of the intestine (villin-Cre^ERtm), combined with a floxed Kras G12D mutation (Kras^G12D/+), Trp53 deletion (Trp53^−/−) and Notch1 intracellular domain (ICD) overexpression (Rosa26^N1icd/+). These mice develop metastases in lymph nodes, lungs and liver [5]. Although the in vivo models are helpful for studying the features of the metastatic process, these are expensive and time-consuming. In addition, the use of in vitro model systems outweighs the use of laboratory animals from an ethical perspective. A faster and simpler way to investigate migration and invasion, one of the first steps to initiate metastasis development, is the transwell in vitro assay, which has been extensively used with cancer cell lines. This assay employs a specialized multi-well plate with two different chambers separated by a membrane. Cells are seeded in the upper chamber, while a chemoattractant is placed in the lower chamber to stimulate the directional cell movement [6].

Despite the fact that cell lines have been instrumental for obtaining better insight in tumor biology and remain a valuable tool, they have some limitations. One of these is their inability to mimic the heterogeneity and cellular diversity typical of tumors. In fact, cell lines often represent a single cell type, making them less than ideal models for studying the interactions between different cell populations that make up cancers [7,8]. In contrast, the development of organoids has revolutionized the study of tissue organization and dynamics and recapitulates the heterogeneity and the 3D structure of tissues. In addition, organoids derived from cancers or, alternatively, organoids transformed in vitro have provided exciting new insights into tumor biology, therapy resistance as well as clonal evolution and are thought to provide a more accurate representation of cancer biology [9]. Thanks to these characteristics, organoids represent a step forward compared to the simpler cell line models and can be used to study the mechanisms of tumor development, providing a platform for the discovery of new drugs [10].

Despite their extensive use, several important features have been more complicated to study in organoids. Although differences between primary and metastatic lesions in CRC have been investigated before, with help of patient-derived organoids, the migratory and invasive capacity of these cells, which could result in metastasis, has been complicated to tackle due to the lack of appropriate methods [11,12,13]. Recently, multiple studies tried to optimize a transwell in vitro assay with use of organoids; however, most of them are time-consuming and focus on a specific cancer type or cell type [14,15,16]. Here, we propose a detailed step-by-step method that allows the study of migration and invasion for mouse intestinal-derived organoids within 3 to 4 days. First optimal cell harvesting was developed for a transformed organoid line that was expected to be migratory. Different chemoattractants were tested to create a gradient that would allow these organoids to migrate. Subsequently, a comparison was made for the migration capacity of mouse intestinal organoids carrying different mutations, commonly found in CRC. After migration, an assay was developed to study invasion. This study provides a reliable method for investigating the migration and invasion of mouse intestinal organoids.

2. Materials and Methods

2.1. Mouse Strains

Four non-induced genetically engineered mouse strains have been used in this study and include combinations of the following genotypes: Apc^fl/fl, Kras^LSL-G12D/+, Trp53^fl/fl, Rosa26^LSL-N1icd/+ and carry a villin-Cre^ERtm construct to undergo in vitro recombination upon tamoxifen addition (see Section 2.2). Animal breeding and isolation of the proximal part of the small intestine were performed in accordance with the UK Home Office license (Project License 70/8646) and were subject to review by the animal welfare and ethical review board of the University of Glasgow.

2.2. Organoid Culture

Organoids were isolated from the proximal part of the small intestine of the mouse, as described previously [17]. Organoids were cultured in Matrigel (356231, Corning Life Science, Amsterdam, The Netherlands) and medium composed of advanced DMEM/F12 (12634010), N2 (17502048) (100× diluted) and B27 (17504044) (50× diluted) supplements, 10 mM HEPES (15630-056), Antibiotic-Antimycotic (15240062, containing: 100 units/mL of penicillin, 100 μg/mL of streptomycin, 0.25 μg/mL Amphotericin B) and 2 mM Glutamax (35050-038) (all Gibco, ThermoFisher Scientific, Bleiswijk, The Netherlands), 50 ng/mL mouse EGF (315-09, ThermoFischer), R-spondin (20 vol% of in-house-produced conditioned medium) and Noggin (10 vol% of in house produced conditioned medium) and maintained in humidified air containing 5% CO₂ at 37 °C. Overnight addition of 4-Hydroxytamoxifen (4-OHT) (1 µM, H7904, Sigma Aldrich, Merck Life Science BV, Amsterdam, The Netherlands) to culture medium was used for in vitro recombination of genes containing loxP sites.

2.3. Migration Assay

Organoids were harvested in cell-recovery solution (CRS, 354253, Corning Life Science BV, Amsterdam, The Netherlands) on ice for 30 min to dissolve the Matrigel and subsequently resuspended in TrypLE (12604013, ThermoFisher Scientific, Bleiswijk, The Netherlands) for 3 min at 37 °C to dissociate into single cells. Cells were counted and 40.000 cells were seeded onto the membrane of the upper chamber (transwell, 8.0 µm pore, 3422 Corning) in 300 µL medium and 500 µL medium including chemoattractant was added to the lower chamber. After 72 h of incubation at 37 °C in humidified air containing 5% CO₂, the lower part of the membrane was fixed with 4% paraformaldehyde (157-8, Electron Microscopy Sciences, Aurion, Wageningen, The Netherlands) for 10 min at room temperature, washed with PBS and non-migrated cells were removed from the upper part of the membrane with a cotton swab. Staining with crystal violet (CV, Merck 548-62-9) was performed for 30 min at room temperature. Inserts were washed with tap water and dried for 24 h upside down before imaging.

2.4. Invasion Assay

Invasion assays were initiated according to the same procedure as described above for the migration assay, except for the use of transwell inserts coated with Matrigel (biocoat Matrigel invasion chamber, 8.0 µm pore, 354480 Corning Life Science BV, Amsterdam, The Netherlands) and hydration of the membrane before use, according to the manufacturer’s instructions. For the 3D invasion assay, uncoated inserts, (transwells, 8.0 µm pore, 3422 Corning) were used and single cells were mixed with 50 µL of Matrigel and seeded on the membrane of the upper chamber. Membranes were fixed and stained after 96 or 144 h.

2.5. Imaging and Quantification of Transwell Migration/Invasion Assay

The fixed and stained organoids were imaged on a Leica DM6 fluorescent microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany). The images were processed and normalized using the Colony Area Plugin provided by ImageJ (1.54j, NIH, Bethesda, MD, USA). A threshold was automatically calculated by the software, which removed most of the background; afterwards, a size threshold was applied using Matlab (R2023b, Natick, MA, USA) to remove the remaining background. The output provided by Matlab was the percentage of area of the membrane covered by cells.

3. Results

3.1. Mouse Intestinal Organoids Migrated in a Gradient-Dependent Manner Towards FCS

To optimize the transwell in vitro migration assay, an organoid model with potential migratory capacity was needed. One good candidate was the previously mentioned KPN model. These genetically modified mice have a tamoxifen-inducible Cre recombinase under the control of the villin promoter, which, upon activation, specifically recombines loxP sites in the epithelial compartment of the intestine. This will lead to KRAS activation, loss of Trp53 and N1-ICD overexpression.

The model resembles the most aggressive CRC subtype found in humans, CMS4, and indeed is able to showcase metastasis in different organs, of which 80% are in the liver [5,18]. Organoids were generated from the small intestine of a non-induced villin-Cre^ERtm KPN mouse. To activate the Cre recombinase in an in vitro setting, cultures were treated with 4-Hydroxytamoxifen (4-OHT), resulting in complete recombination of the different floxed loci. Before starting the transwell assay, Matrigel, in which the cells were cultured, was dissolved, with cell recovery solution (CRS) to avoid any basement membrane presence during the experiment. Then, organoids were dissociated with TrypLE, counted and seeded as single cells onto the membrane of the insert in the upper chamber. After 3 days, the part of the membrane of the lower chamber was fixed, and non-migrated cells on the upper part of the membrane were removed with a cotton swab. Staining with crystal violet (CV) dye was performed to visualize migrated cells (Figure 1).

To test this transwell assay, in vitro recombined KPN organoids were placed in the upper chamber of a transwell. In order to create a gradient, FCS, placed in the lower chamber, is normally used as chemoattractant (Figure 2A). In organoid cultures, FCS, which is typically used for cell line cultures, is replaced by two supplements that contain essential additives for FCS-free cultures: B27 and N2 [19]. To find the best chemoattractant for organoids migration, two chemoattractants were tested in comparison to a negative control. In the upper chamber, KPN organoids were resuspended in organoid medium, supplemented with EGF, Noggin and R-spondin (ENR), but without B27 and N2 supplements to mimic FCS starvation, as routinely used for the cell line migration assays. In the negative control condition, the exact same media composition as that used for the upper (ENR medium without B27 and N2) chamber was used for the lower chamber. For the other two conditions, chemoattractants were added to stimulate migration. In the first setting, ENR with B27 and N2 was added to the lower chamber, while in the second setting, ENR was supplemented with 10% FCS (Figure 2B). In the control setting, KPN cells showed limited migration. Similarly, when B27 and N2 were used as chemoattractants, no significant change in the level of migration was detected. In contrast migration of KPN organoids was significantly stimulated by FCS (Figure 2C,D). As previously mentioned, organoids are normally grown in a medium supplemented with B27 and N2, and since the experiment demonstrated that migration was induced by FCS, we tested whether seeding organoids in ENR + B27/N2 in the upper chamber and adding B27/N2 to the FCS-containing lower chamber would further improve the migration percentage of KPN organoids (Figure S1A). This condition rather dampened the migration capacity of KPN organoids (Figure S1B,C), which points to the possibility that migration in this classical transwell assay may have previously been overlooked due to a choice of medium.

3.2. KPN Organoids Migrated More Extensively than APC, K and KP Organoids

To determine whether migration is a typical feature of transformed organoids or is unique to the aggressive KPN organoid model, other genetically modified mouse organoids were used. In vitro organoid cultures derived from non-induced villin-Cre^ERtm Apc^fl/fl (APC), villin-Cre^ERtm Kras^LSL-G12D/+ (K) and villin-Cre^ERtm Kras^LSL-G12D/+ plus Trp53^fl/fl (KP) mice were included and mutations were activated after in vitro recombination with 4-OHT. Alterations in these genes are frequently detected in CRC and thus, we are interested in determining their impact on migration. The KP organoid model is of particular interest, as in vivo activation of recombination leads to the development of primary tumors with the same speed as in the KPN mouse model; however, KP mice do not develop metastases [5]. Interestingly, in our newly developed organoid transwell migration set-up, APC organoids showed only marginal migration, while K and KP organoids did not migrate at all compared to KPN organoids (Figure 3A,B). This was not due to a lack of attachment and survival, as all lines were able to grow and expand without being harmed by supplement starvation and the 2D growth conditions (Figure S2). This provides strong evidence that transwells can be used for migration analysis of organoids as long as the right genotype, medium composition and attractant are used.

3.3. KPN Organoids Invaded Matrigel-Coated Transwells

The ability of cells to invade through the extracellular matrix (ECM) is a crucial step in the metastatic cascade. Therefore, our migration protocol was adapted to also test invasive capacity by using Matrigel-coated transwell inserts (Figure 4A). After 3 days, KPN cells were unable to significantly invade through the Matrigel layer. However, on day 4, significant levels of invasion were detected, indicating that the cells require more time to pass through the Matrigel layer as compared to the direct migration assays (Figure 4B,C). Furthermore, to mimic the normal growth conditions, the invasive capacity of organoids grown in 3D, using a thicker layer of Matrigel, was investigated. We compared the efficiency of our 2D pre-coated Matrigel invasion transwell insert to a transwell insert, which we coated with 50 µL Matrigel including organoids (as single cells) resuspended within the Matrigel at moment of seeding. After 4 days, very few cells invaded the filter. However, after prolonging the assay to 7 days, cells passed through the Matrigel and filter, although less effectively compared to the coated Matrigel filters (Figure S3). This lower efficacy of invasion might be due to the fact that these KPN cells started to grow into cystic organoid structures when seeded in the layer of Matrigel, which likely restricts the invasive properties of the cells.

4. Discussion

This study describes an optimized assay to determine the migratory and invasive behavior of organoid lines in an in vitro setting. A migration or invasion assay is a quicker way to investigate the processes underlying metastasis initiation compared to in vivo experiments with mice. However, despite the fact that examples of migration exist [14,15,16], a general migration or invasion method using mouse organoids has not been described in the literature before. Therefore, we optimized a known transwell assay to find the settings, such as chemoattractant and medium conditions, that allow cells derived from organoids to migrate and/or invade. The use of mouse- or human cancer-derived organoids can avoid limitations of cell lines that are grown in 2D. The organoids used in this study are derived from the small intestine of non-induced genetically engineered mouse models and used as in vitro cultures within Matrigel to subsequently introduce mutations/deletions and subsequent transformation via loxP-dependent recombination. After testing two distinct chemoattractant conditions, classical FCS proved to be the most effective inducer of migration in KPN organoids. As an addition, we want to emphasize that the composition of medium has an impact on migratory capacity, as shown in Figure S1. Furthermore, we compared the migratory potential of several organoid models carrying different mutations. KPN turned out to migrate more than A, K and KP organoids, which are largely deficient for directed migration.

We also optimized the invasion assay, which differs from the migration assay through the Matrigel-coated membrane. Compared to the migration assay, 3 days was insufficient for the cells to invade, while at 4 days invasion was evident. Timing should therefore be evaluated carefully to avoid false-negative results.

In order to use this assay for human organoids and mouse organoids derived from different organs, further optimization should be performed. It is important to look into different chemoattractants and culture settings to mimic starvation and create a gradient as has been widely used in transwell assays with cell lines. This paper provides a reliable starting condition to analyze the migratory and invasive properties of organoids and study the biology that is crucial for metastasis.