Systematic comparative protein expression profiling of clear cell renal cell carcinoma: a pilot study based on the separation of tissue specimens by two-dimensional gel electrophoresis

Abstract

Proteome-based technologies represent powerful tools for the analysis of protein expression profiles, including the identification of potential cancer candidate biomarkers. Thus, here we provide a comprehensive protein expression map for clear cell renal cell carcinoma established by systematic comparative two-dimensional gel electrophoresis-based protein expression profiling of 16 paired tissue systems comprising clear cell renal cell carcinoma lesions and corresponding tumor-adjacent renal epithelium using overlapping narrow pH gradients. This approach led to the mapping of 348 distinct spots corresponding to 248 different protein identities. By implementing restriction criteria concerning their detection frequency and overall regulation mode, 28 up- and 56 down-regulated single target spots were considered as potential candidate biomarkers. Based on their gene ontology information, these differentially expressed proteins were classified into distinct functional groups and according to their cellular distribution. Moreover, three representative members of this group, namely calbindin, gelsolin, and heart fatty acid-binding protein, were selected, and their expression pattern was analyzed by immunohistochemistry using tissue microarrays. Thus, this pilot study provides a significant update of the current renal cell carcinoma map and defines a number of differentially expressed proteins, but both their potential as candidate biomarkers and clinical relevance has to be further explored in tissues and for body fluids like serum and urine.

Fig. 1.

Consensus master maps of differentially expressed proteins in ccRCC lesions compared with tumor-adjacent renal epithelium. Representative 2DE gels displaying the consensus protein expression profiles characteristic for ccRCC using pH 4–7 (A) and pH 6–11 (B) gradients are shown. The numbers assigned to the mapped protein spots correspond to the proteins listed in Table II and supplemental Table 1.

Fig. 2.

Classification of differentially expressed proteins according to their cellular function and their cellular localization. The pie charts display the classification of the subset of differentially expressed proteins listed in Table II into 12 functional categories (A) and into 10 distinct cellular compartments (B). The pool of proteins comprises 84 distinct protein identities defining the 123 spots, which were identified in at least five independent biopsy systems. The distribution frequencies in regard to the specified categories within the given chart pie are indicated in percentage of the total number of entries. For each pie chart a cutoff value was set to 5%, meaning that all of the cellular functions/compartments represented below this threshold were summarized under other.

Fig. 3.

Immunohistochemical analysis for calbindin expression in tumor-adjacent renal epithelium and distinct kidney tumor subtypes. Randomly selected sections of the TMA representing tumor-adjacent renal epithelium and various RCC subtypes were used to document the immunohistochemical staining with the anti-human calbindin-specific mAb. A shows the immunohistochemical analysis of calbindin in tumor-adjacent kidney tissue at 200× magnification; B refers to the staining pattern at a magnification of 400×. Calbindin is expressed in some but not all cells of the proximal (solid arrows) as well as the distal tubule system (open arrows) in non-tumorous kidney tissues. Yet both stainings show sporadic strong positive stainings of the epithelium of both the proximal and the distal tubule systems. In contrast, the ccRCCs (C; magnification, 400×) and RCC of the chromophobic type (D; magnification, 400×) lack calbindin staining.

Fig. 4.

Immunohistochemical analysis for calbindin expression using TMA technology. Immunohistochemical staining of the TMA representing the four major RCC subtypes along with corresponding tumor-adjacent renal epithelium was performed using a human anti-calbindin-specific mAb (TMA panel: 40 ccRCC lesions, 31 chromophilic RCC lesions, 16 chromophobic lesions, and nine oncocytic lesions, each flanked by corresponding tumor-adjacent renal tissue sections). The classification of the staining pattern indicated in each panel is based on the criteria defined under “Experimental Procedures.” Black segments indicate strong positive staining, dark gray segments indicate intermediate staining intensity, light gray segments indicate weak staining intensity, and white segments indicate negative staining. The data are expressed as relative percentage of total samples stained. The staining pattern for calbindin is shown on ccRCC (A), chromophilic RCC (B), chromophobic RCC (C), and renal cell adenomas of oncocytic type (D).

Fig. 5.

Immunohistochemical analysis for gelsolin expression in tumor-adjacent renal epithelium and distinct kidney tumor subtypes. Randomly selected sections of the TMA representing tumor-adjacent renal epithelium and RCC subtypes were used to document the immunohistochemical staining with the anti-human gelsolin-specific mAb. In tumor-adjacent kidney tissues (A), expression of gelsolin is mainly observed in the epithelium of the distal tubule system and collecting ducts, whereas the epithelium of the proximal tubule system as well as the glomeruli (despite not being represented on this section) stained negative. More than 30% of the ccRCCs lack gelsolin staining (B). In contrast, tumors originating from the distal tubule system/collecting ducts like chromophobic RCC (C) and from benign renal adenoma of the oncocytic type (D) displayed strong or intermediate positive staining for gelsolin in more than 75% (C) and 55% (D) of all cases analyzed. Magnification in A–D, 400×.

Fig. 6.

Immunohistochemical analysis for gelsolin expression using TMA technology. The immunohistochemical staining pattern for gelsolin using the RCC-specific TMA consisting of ccRCC (A), chromophilic RCC (B), chromophobic RCC (C), and renal cell adenomas of the oncocytic type (D) are shown. The composition of the TMA panel, classification of the staining pattern, and color code are the same as in Fig. 4.

Fig. 7.

Immunhistochemical analysis for H-FABP expression in tumor-adjacent renal epithelium and distinct kidney tumor subtypes. Randomly selected sections of the TMA representing tumor-adjacent renal epithelium and RCC subtypes were used for immunohistochemical staining with the anti-human H-FABP-specific polyclonal Ab. In tumor-adjacent kidney tissue, the epithelium of the proximal and distal tubule systems as well as mesangial cells show strong cytoplasmic staining for H-FABP (A). RCCs of the clear cell subtype (B) demonstrate strong positive cytoplasmic staining in only 23% of all tumors tested, whereas more than 60% of chromophilic RCCs (C) were negative for H-FABP. In contrast, all benign oncocytomas (D) exhibit a strong positive cytoplasmic staining for H-FABP. Magnification in A–D, 200×.

Fig. 8.

Immunohistochemical analysis for H-FABP expression using TMA technology. Immunohistochemical staining of the TMA representing the four major RCC subtypes along with corresponding tumor-adjacent renal epithelium is shown. The composition of the TMA panel in this analysis was slightly modified by representing just 26 ccRCC lesions. Besides that, the classification of the staining pattern and the color code are the same as those in Fig. 4. The frequency of the different staining patterns for H-FABP on sections representing ccRCC (A), chromophilic RCC (B), chromophobic RCC (C), and adenomas of oncocytic type (D) are shown.