Several studies, including some looking at renal cancers, point to stromal components as key players in inducing tumorigenesis and in the acquisition of invasive behaviors 
. Hence, investigators have suggested that analysis of the tumor stroma may render meaningful prognostic data 
. In this context, targeting tumor-associated stroma may hamper its induced permissiveness towards epithelial cell transformation and/or invasion and, consequently, increase the efficacy of chemo- and radiotherapies 
. It is well established that tumor activated fibroblast express myofibroblastic markers, such as α-SMA 
, palladin 
and EDA-fibronectin 
. As the role of these stromal markers in RCC progression is currently unknown, we investigated the association between the expression of these markers and RCC prognosis.
In a recent collaborative work, we demonstrated that increased expression of stromal palladin can be found in fibroblasts associated with pancreatic ductal adenocarcinoma and other neoplasias such as lung, skin, breast and kidney 
. Therefore, and as previously suggested 
, one could speculate that palladin expression may alter some key stromal properties that induce or increase tumorigenesis. To this end after analyzing our TMA data, we concluded that stromal expression of palladin correlated with the survival of RCC patients; higher levels of stromal predicted lower overall survival times. Hence, palladin may constitute an independent marker for prognosis. Strikingly, the predictive value of this stromal marker was highly significant in patients who had not developed metastasis suggesting that it could constitute a high risk marker in this patient population. Another stromal marker whose expression correlated with stromal activation, α-SMA, showed a rather high background staining in the stroma associated to normal kidney tissue resulting in less impressive differences with the stroma associated with tumors. In addition, the reduced number of samples for which survival data are available decreased the statistical significance between the mean staining score between these two populations. In this context, palladin seemed to be a stronger predictor of survival than α-SMA since the correlation showed by comparing palladin staining in stroma associated with normal or tumoral tissue remained statistically significant in spite of the few samples whose survival data were not available.
The expression of markers suggestive of stromal activation could serve as indicators of tumor-stromal interactions 
, a largely understudied aspect of RCC. Palladin plays important roles in actin organization, sarcomere integrity, and cytoskeleton architecture. Palladin has also been associated with stimulation of cell motility and increased matrix stiffness, both characteristics attributed to tumor-associated activated stroma 
. To address changes associated with the RCC stroma, we developed a novel renal fibroblast derived 3D culture system that, similar to other systems 
, effectively mimics many aspects of the in vivo
stromal microenvironments. We used fibroblasts harvested from five late RCC stage cases with matching non-activated (i.e., normal), primary and metastatic tumors and one localized early stage case as a control. We observed that self-derived 3D cultures recapitulate the original (i.e., in vivo
) expression levels of the four selected markers. Moreover, 3D matrix-induced α-SMA and palladin in vitro
levels greatly correlated with the stromal activation of fibroblasts (myofibroblasts). On the other hand, uPARAP and EDA-fibronectin expressions appeared to be regulated independently of 3D ECMs suggesting that perhaps their expression depends on additional parameters such as tissue type or cellular location. Previous reports from our laboratory indicated that, in order to achieve in vivo
-like stromal expression patterns, culturing cells onto fibroblast-derived matrices or as un-extracted 3D cultures, is often necessary 
. By the use of these 3D cultures, we demonstrated that levels of stromal markers α-SMA, palladin and, to some extent, uPARAP are consistently and significantly increased compared to those of all harvested from normal kidney tissues, reflecting their usefulness as surrogates of in vivo
specimens. On the other hand, the fibronectin spliced form EDA did not show significant variations.
In spite of the clear differences in the levels of expression of α-SMA and palladin observed when comparing 3D cultures produced by tumor-derived vs. normal kidney-derived fibroblasts, relatively high distributions among samples were also observed. To this end, it has been suggested that myofibroblastic activation occurs in progressive phases 
and that perhaps tumor-associated stroma can also be sorted into various progressive stages 
. In an effort to determine whether incorporating stromal features into an enhanced staging scheme could be used for RCCs, we investigated whether stroma progression accompanies or rather develops at an independent pace from tumor staging. We questioned if our 3D culturing system effectively mimics the original in vivo
stromal features. Individual analysis of RCC cases indicated that levels of stromal markers do not necessarily always correlate with the original stages of the tumors. Nevertheless, RCC cases showing the greatest expression levels of both α-SMA and palladin indeed correlated with the most activated 3D cultures sorted using our published methods 
. Interestingly, the same cases sorted as ‘activated’ presented in vivo
characteristics of activated stroma such as increased levels of stromal markers palladin and α-SMA indicating that our 3D system indeed mimics in vivo
Our results suggest that the rate of stroma transformation does not correlate with the tumor stage assigned by the evaluation of the tumor cells proposing the possibility that the assessment of stroma progression may complement tumor stage as a clinical prognostic variable in RCC. Moreover, we believe that we have identified stromal palladin as an early risk factor predictor for RCCs.