Loss of pVHL function, characteristic for clear-cell renal cell carcinoma (ccRCC), causes increased expression of CXCR4 chemokine receptor, which triggers expression of metastasis-associated MMP2/MMP9 in different human cancers. The impact of pVHL on MMP2/MMP9 expression and their relationship to CXCR4 and its ligand CXCL12 in ccRCC is unclear. By using reverse transcription PCR, immunofluorescence and immunohistochemistry, strong mRNA and protein expression of CXCR4, CXCL12, MMP2, MMP9 and MMP inhibitors TIMP1 and TIMP2 was found in VHL-null 786-O ccRCC cells. Loss of CXCR4/CXCL12 expression after restoration of VHL function in these cells was accompanied by a significant reduction of MMP2 and MMP9 expression, whereas neither TIMP1 nor TIMP2 expression was affected. Using real-time PCR analysis, higher MMP2 (p = 0.0134) and MMP9 (p = 0.067) mRNA expression levels were detected in primary ccRCC with strong CXCR4 compared to cases with weak CXCR4 expression. There was no association between CXCR4 and TIMP1 or TIMP2 mRNA expression. MMP2 protein expression data obtained by immunohistochemistry on a tissue microarray uncovered positive cytoplasmic staining in 290/380 (76%) primary ccRCCs. Co-expression of CXCR4 and MMP2 was found in 282 of these tumours (74%). Our in vitro and in vivo data strongly indicate that pVHL coordinately regulates expression of metastasis-associated genes CXCR4/CXCL12 and MMP2/MMP9 but the exact molecular mechanism of this regulation remains to be determined. Co-expression of CXCR4 and CXCL12, as demonstrated in VHL-null 786-O cells, might enable ccRCC progression and metastatic dissemination by autocrine receptor stimulation, even in the absence of exogenous CXCL12.

Different methods for snap freezing surgical human tissue specimens exist. At pathology institutes with higher work loads, solid carbon dioxide, freezing sprays, and cryostat freezing are commonly used as coolants for diagnosing frozen tissue sections, whereas for tissue banking, liquid nitrogen or isopentane cooled with liquid nitrogen is preferred. Freezing tissues for diagnostic and research purposes are therefore often time consuming, laborious, even hazardous, and not user friendly. In tissue banks, frozen tissue samples are stored in cryovials, capsules, cryomolds, or cryocassettes. Tissues are additionally embedded using freezing media or wrapped in plastic bags or aluminum foils to prevent desiccation. The latter method aggravates enormously further tissue handling and processing. Here, we describe an isopentane-based workflow which concurrently facilitates tissue freezing and processing for both routine intra-operative frozen section and tissue banking and satisfies the qualitative demands of pathologists, cancer researchers, laboratory technicians, and tissue bankers.