Mesenchymal stem cells promote metastasis through activation of an ABL-MMP9 signaling axis in lung cancer cells

Mesenchymal stem cells (MSCs) are recruited and activated by solid tumors and play a role in tumor progression and metastasis. Here we show that MSCs promote metastasis in a panel of non-small cell lung cancer (NSCLC) cells. MSCs elicit transcriptional alterations in lung cancer cells leading to increased expression of factors implicated in the epithelial-to-mesenchymal transition (EMT) and secreted proteins including matrix metalloproteinase-9 (MMP9). MSCs enhance secretion of enzymatically active MMP9 in a panel of lung adenocarcinoma cells. High expression of MMP9 is linked to low survival rates in lung adenocarcinoma patients. Notably, we found that ABL tyrosine kinases are activated in MSC-primed lung cancer cells and functional ABL kinases are required for MSC-induced MMP9 expression, secretion and proteolytic activity. Importantly, ABL kinases are required for MSC-induced NSCLC metastasis. These data reveal an actionable target for inhibiting MSC-induced metastatic activity of lung adenocarcinoma cells through disruption of an ABL kinase-MMP9 signaling axis activated in MSC-primed lung cancer cells.

MMP7 transcript expression was also significantly increased following MSC/NSCLC co-culture ( Figure  3A). The authors should clarify why only MMP9 was pursued.
RESPONSE: Whereas MMP9 expression was significantly increased in the PC9 lung cancer cells compared to MSCs following MSC/NSCLC co-culture, increased MMP7 expression following co-culture was predominantly detected in MSC cells and to a much lesser extent in the PC9 lung cancer cells. We further confirmed the findings in Figure 3A by directly comparing expression of MMP7 to MMP9 after co-culture following FACS sorting of PC9 and H1650 lung cancer cells and analysis of mRNA and protein expression as shown in Supplemental Figures 4B-C. Thus, we focused on MMP9 because this protease was consistently increased in the cancer cells following MSC/NSCLC co-culture.
In Figure 4A and B, pCrkL and pSTAT3 are used as readouts for ABL kinase activity, but only pCrkL is presented in Figure 4C and D to validate the effect of treatment with GNF5 or ABL001. Levels of pSTAT3 should be added to these panels for consistency if possible.

RESPONSE:
We have performed p-Stat3, total Stat3 and total CrkL western blot analysis as requested and have added the new data to Figure 4C and 4D.
Line 297 refers to "Affimetrix." Please check whether this should be "Affymetrix." RESPONSE: We have corrected Affimetrix to Affymetrix.
In contrast to the PC9 cells ( Figure 7C), the impact of ABL depletion on survival in HCC827 is not mentioned. Even if not significant, the data for HCC827 could be included for completion, and the authors could discuss potential reasons for the difference.
RESPONSE: HCC827 cells are larger in size than PC9 cells and aggregate in clusters that can elicit vessel obstruction and rapid death in mice injected with the same injection protocol as that used for PC9 cell injection. Therefore, we injected a reduced number of HCC827 cells (1x10 5 ) by intracardiac route to avoid blood vessel clotting that often led to death of mice. Furthermore, the doubling rate of HCC827 cells is significantly slower than PC9 cells. Therefore, injection of 1x10 5 HCC827 cells in mice would necessitate a much longer timeline for the mouse survival studies which is estimated would be 9 to 12 months. Given the excessive extended timeline we were not able to carry out this experiment with HCC827 cells.
Please specify that the bone marrow MSCs from Lonza are human origin in the Materials and Methods.
RESPONSE: The bone marrow derived MSCs are human origin. We have added this information to the "Materials and Methods" section.

Reviewer #2
We thank the reviewer for stating that the manuscript "is a very interesting report of ABL kinase modulating EGFR-mutant non-small cell lung cancer (NSCLC) invasiveness and metastatic colonization through metalloproteinase (MMP) upregulation", and that the "work is well designed, well performed and I believe it will appeal to PlosOne audience". We have addressed the minor suggestions below:

-BM-MSC tropism to inflammatory and cancer sites is well reported. However, the exactly extent that it could happen in pathophysiological conditions it is not known. The work shows a correlation of high MMP9 expression, which is upregulated by stromal BM-MSC, and poor patient outcome. Have authors assessed how many of these patients have significant tumor stroma associated-lung adenocarcinoma?
RESPONSE: We thank the author for this comment and agree that it would be of interest to more fully elucidate how tumor stroma infiltration correlates with patient outcomes in lung adenocarcinoma.
Unfortunately, the patient survival data showing a correlation of high MMP9 mRNA expression with poor survival outcomes is not amenable for such interrogation. As described in the methods section, these data were analyzed using the KMPlot analysis tool originally developed by Győrffy et al, which houses a collection of patient datasets linking tumor gene expression (microarray) with survival outcomes (1). As the microarray data used in these datasets does not provide corresponding information on stromal involvement (for example, through IHC or H&E staining/imaging), we are unable to determine from this particular dataset if patients with high tumor MMP9 expression present with increased stromal infiltration.
A few recent reports have sought to explore the relationship between tumor vs stroma (primarily through H&E tissue staining/analysis) in the context of patient survival. For example, Costas et al used immunohistochemistry to analyze stromal involvement in a cohort of NSCLC patients with available survival data and found that the presence of fibrotic stroma correlated with increased mortality (2). Zhang and colleagues, using a similar approach in a larger cohort of 404 NSCLC patients, defined a measure termed the "tumor-stroma ratio" (TSR) and found that stroma-high tumors were associated with an increased risk of relapse following surgical resection and poor prognosis compared to patient tumors with minimal stromal infiltration (3). These results are further supported by a more recent study of TSR in NSCLC patients, which came to similar conclusions (4). While these human patient data do not directly examine tumor MMP9 expression in the context of stromal recruitment to tumors, at least one study identified a correlative relationship between tumor MMP9 expression and increased stromal involvement in NSCLC (5). As most of these studies are largely correlative, we agree with the reviewer that future studies in this area which are beyond the scope of this work, should be undertaken to evaluate a causal relationship between patient survival and stromal involvement. Moreover, in order to ascertain which cell type is responsible for increased MMP9 expression in patient specimens, it would be necessary to use single cell RNA-seq as current databases do not indicate whether increased MMP9 expression is associated with lung tumor cells, MSCs, immune cells, or other cell types in the tumor microenvironment.