Bulk tumour cell migration in lung carcinomas might be more common than epithelial-mesenchymal transition and be differently regulated

Migration and invasion into the stroma is a requisite for cancer metastasis. Two mechanisms have been described in the last decade, namely epithelial to mesenchymal transition (EMT) and complex tumour cell migration. In EMT, as primarily described in experimental studies, the tumour cells downregulate adherence proteins, lose contact to cells, and change to a mesenchymal phenotype, often by exchanging keratin for vimentin or α-actin [1, 2]. These cells develop invadipodia and move as individual cells or in small groups. Several genes have been identified as EMT drivers, namely snail family transcriptional repressor 2 (Slug), twist family bHLH transcription factor 1 (TWIST), Zinc finger E-box binding homeobox 1 (ZEB1), nuclear-translocated β-Catenin, transforming growth factor beta (TGF-β), and the frizzled class receptor family (frizzled) [3‐10]. Among examples for EMT in pulmonary carcinomas are small cell carcinomas (SCLC), micropapillary adenocarcinomas, and pleomorphic carcinomas (PC), whereas in the majority of adenocarcinomas (AC), squamous cell carcinomas (SCC), and large cell carcinomas (LC), EMT seems to be rare. In complex tumour migration, the cells form cell clusters and disconnect from the main tumour [11]. EMT has been thought to be essential for metastasis. However, recent findings have challenged this view and proposed complex tumour cell migration as an alternative. Some have called this hybrid EMT, and it has even been experimentally shown that cells can metastasize without changing to a mesenchymal phenotype, called the epithelial migration type [12‐15]. Although SCLC as well as micropapillary adenocarcinomas move in small cell clusters of 3–5 cells or as single cells and invade the primary as well as the metastatic organ site diffusely, in the majority of cases, they do not lose their epithelial phenotype. Neither loses cytokeratin – thus, they are a part of hybrid-EMT. Pulmonary sarcomatoid carcinomas, especially spindle cell carcinomas, often occur in the primary tumour with a mesenchymal phenotype, expressing α-actin instead of keratin. They may continue doing this during metastasis, or they may revert back to an epithelial phenotype at the metastatic site.

Due to the fact that vascular invasion is a poor prognostic factor [16], we usually evaluate carcinomas for vascular invasion. We identified well-formed AC and SCC cell clusters within the vascular walls, still forming differentiated structures such as glands or plate-like sheets. Because these tumour cells are disconnected from the main tumour, they must have migrated in these complexes or bulks (Fig. 1a, b, c). These bulks do not lose their keratin cytoskeleton and generally do not express vimentin or other markers of EMT, indicating that bulk cell migration is an alternative mechanism, for which the driving genes have not been identified. In Drosophila wing border cell migration, four key genes have been identified as being related to this complex cell movement: receptor of activated C kinase (Rack1), brinker (Brk), mother against dpp (Mad), and saxophone (SAX) [17].

As a first step, we aimed to explore the frequency of bulk migration in AC and SCC, to evaluate molecules which might be associated with migration, invasion, and metastasis using immunohistochemistry. We are aware of limitations of this study: morphology provides a snapshot at a certain stage of development of a carcinoma, but cannot provide insight into dynamics of migration. However, we can compare expression patterns in the primary tumour with migrating and metastatic cells, which is not possible in a cell culture system. A morphological analysis can provide necessary information on which to base an experimental design.

Thirty cases of AC and SCC were retrieved from the Lung Archive, all characterized by vascular invasion into pulmonary arteries or veins. The selected cases required having the central tumour, the invasion front to lung or pleura, and the vascular invasion on the same tissue slide to compare staining patterns side by side (Fig. 1; SETstudy). With a literature search, several molecules were identified as being associated with migration and invasion. Antibodies for these molecules were purchased and tested for their specificity. Dilution and pre-treatment was adapted when necessary (for details, see Table 1). As the study set had a rather small number of cases, we aimed to validate the importance of the markers in a larger cohort of NSCLC: tissue microarrays (TMA) were chosen which contained a total of 325 AC, 142 SCC, and 61 LC (validation set for immunohistochemistry; TMAval); out of these cases, whole tissue sections from 115 cases of SCC and 117 cases of AC were randomly chosen for the evaluation of bulk and/or EMT migration (SETval). To evaluate the importance of the markers for metastasis, another TMA, composed of 45 cases of primary tumour and up to three corresponding metastasis (TMAmets), was chosen.

Each case in the TMA was represented by at least 3 cores of tumour tissue and 1 core of normal lung tissue or metastatic site. Immunohistochemistry in TMAval/TMAmets was performed for those markers, which showed a different staining pattern in the study set between the central tumour and the two invasion sites (front and/or vascular invasion). Three pathologists (MZ, LB, HP) evaluated the slides independently. All cases were discussed and re-evaluated together in cases of discordant scores.

Migration of carcinoma cells is a complex process which requires modification of the adhesion to their neighbouring cells, formation of invadipodia, disconnection from the primary tumour, development of sensory capacity for oxygen tension and pH gradient, reading adhesion molecules present on matrix proteins to gain orientation (fibronectin, collagens, etc.), and creating energy for movement – all depending on the initiation of a multi-layered genetic program.

Here, we describe bulk cancer cell migration, also called complex movement or hybrid EMT [13, 18], as the predominant mode of migration in pulmonary squamous and adenocarcinomas. Carcinomas even retained their acinar, plate-like, or sheet formation. Our findings are based on a morphological analysis which provides a comparison of the primary tumour, migrating cells, and metastasis. If carcinoma complexes are seen within the vascular wall and lumen, it is very likely that the cells migrated there in the same bulk. Vascular invasion is therefore the optimal site for investigating the mode of migration. A comparison of expression of molecules between the primary tumour and the bulk cells might provide a first insight into which of these factors might be responsible for migration and allow a hypothesis to be formed. Dynamics of this process, such as up- and downregulation of molecules involved in bulk migration, cannot be evaluated on histology, but our findings can be used to create an experimental model.

In conclusion, we identified bulk cancer cell migration as the predominant form in AC and SCC, whereas pure EMT is rare. In addition, we identified a mixed form composed of EMT and bulk migrations which accounts for approximately 25% of AC and SCC. Several proteins known from EMT studies have been identified, but are differently regulated in bulk migration. Twist, vimentin, fascin, Mad, Brk, Tsk5, Rab40B, ERK1/2 and PLCγ are involved in bulk cancer cell migration, but they are not under the rule of TGFβ1 (Additional file 3: Figure S1 show a schematic drawing how the different proteins might interact). The known pathways Wnt-frizzeld-βCatenin and TGFβ1, along with Snail, Slug, and ZEB1, are not involved in bulk cell migration. Bulk, complex, or hybrid migration, is placed in between EMT and epithelial complex migration representing the opposite poles [15, 48]. The homing and metastasis function of Connexin43, CXCR1, and CXCR4 require further study. Bulk migration requires an orchestrated activation of proteins to keep the cells bound to each other and to coordinate the movement. As this study was based on a retrospective analysis of lung carcinoma tissues, no functional assays were done. However, we will explore this type of migration using an in vivo experimental system.