In this study, we successfully established two ccRCC cell lines from two Chinese patients with ccRCC. Up to now, the two cell lines have been maintained in our laboratory for 6 years and cultured for more than 100 passages. The two patients were comparable on the aspects of race, sex, and age at onset of primary ccRCC. Although the microenvironment plays an important role in evolutionary process of the metastatic cells from their primary tumors, the selected metastatic cancer cells maintain their characteristics following long-term in vitro cultures. In vivo study demonstrated that MRCC cells exhibited more malignant and metastatic potential than NRCC (Table 2). Therefore, the differences in cellular and subcellular morphology, cell growth/invasion ability, cytogenetics, cell markers, and expression pattern of metastasis-associated molecules between the two cell lines can designate, at least partially, some important cellular and molecular events related to ccRCC metastasis.
The current study characterized the cell lines of 50–60 generations. It was found that MRCC grew a little slower but exhibited stronger anchorage-independent growth potential than NRCC in vitro. The invasion study indicated MRCC had higher invasion potential than NRCC. Analysis of cell cycle at the same pace of proliferation suggested that MRCC were more frequent in S phase whereas NRCC displayed a typical sub-G1 peak. Thus, compared to NRCC, MRCC exhibited “low proliferation-high invasion-low apoptosis” cell kinetic profile. This is probably related to a large number of glycogen particles stored in the cytoplasm. The “glassy” cytoplasm appearance of ccRCC might be due to glycogen and sterol storage caused by abnormalities in carbohydrate and lipid metabolism [9, 11]. GSK3 is a protein kinase that phosphorylates and inactivates glycogen synthase, the final enzyme of glycogen biosynthesis . In this study, we found that GSK3β, an important member of GSK3 family, was higher expressed in NRCC than in MRCC (Figure 6). The level of GSK3β may be reversely related to glycogen storage in RCC cells. Glycogen-rich carcinomas of clear cell subtype are usually characterized by a peculiar “low proliferation-low apoptosis” cell kinetic profile and associated with cancer aggressiveness [11, 13]. Thus, the level of GSK3β might be reversely related to ccRCC metastasis.
Loss of chromosomal materials on 3p, 8p, 9p, and 14q has been documented in 96%, 22%, 33%, and 41% of ccRCC cases, respectively . The von Hippel Lindau (VHL) tumor suppressor gene on chromosome 3p and stabilization of HIF1α due to loss of VHL function has been shown to be central to development of ccRCC . However, the cytogenetic abnormalities on chromosomes 3p and the difference in HIF1α levels were not found in this study. With the use of single-cell exome sequencing, AHNAK, LRRK2, SRGAP3, and USP6 have been found to be the key mutated genes in the ccRCC patient without VHL mutations . In this study, although we found some differences in their expression patterns (Figure 6), it is hard to interpret the role of the 4 genes in ccRCC in terms of the expression patterns. We found that gains of chromosomes and some abnormal structures were the major chromosomal abnormalities in the two cell strains. Thus, our findings add novel information to the cytogenetic abnormality of ccRCC with different metastatic potentials and make the cell lines good tools to study RCC without VHL mutations.
Our cytometry assay revealed that the two cell lines were positive for CD44 but negative for CD133, CD105, and CD74. CD44 is a hyaluronic acid receptor whose mRNA levels in tumors can distinguish between RCC subtypes and RCC subtypes from oncocytoma and predict RCC metastasis . Unexpectedly, the two cell lines were negative for CD105, a possible marker of ccRCC-initiation cells . Recent studies have confirmed that CD133 is not detectable in RCC cells and tissues [10, 18]. Here we found CD24 positivity was more frequent in MRCC than in NRCC and the same was true for CD56 (Figure 4). CD24 is a cancer stem-like cell biomarker whose expression in tumors is associated with malignant phenotype and poor prognosis of ccRCC and other cancers [19, 20]. CD56 has been found to be expressed in 15%-18% of ccRCC and associated with poor outcome . Although no markers, single or combined, could be defined unequivocally to specifically identify cancer stem cells in a given solid tumor so far , our data indicate that CD24-positive subpopulation might be the most likely stem-like cells that are related to ccRCC metastasis. NRCC and MRCC are epithelial-origin, but MRCC tends to be more mesenchymal-like (Figure 4). Acquisition of mesenchymal properties by epithelial cells, a process called epithelial-mesenchymal transition (EMT), can partially explain the metastatic potential of MRCC.
Although TNFα, VEGF, IL-6 and other cytokine/chemokine from lymphocytes, endothelial cells and mesenchymal cells within the microenvironment are necessary to maintain cancer “stemness” , the expression of these factors in cancer cells is important in maintaining their invasive and metastatic potential. In this study, we found that the transcriptional levels of IL-6, VEGF, HIF2α, TNFα, MMP2, and RhoC were higher in MRCC than in NRCC. Expression of VEGF, MMP2, and RhoC in ccRCC is associated with metastasis and poor prognosis or used to evaluate the effectiveness of therapies on metastatic RCC [24–26]. The expression of IL-6 and TNFα is significantly elevated in high malignant RCC cells compared to low malignant RCC cells . Furthermore, plasma levels of TNFα and IL-6 are associated with poor survival of RCC patients . Interestingly, the expression of HIF2α, rather than HIF1α, was significantly elevated in MRCC than in NRCC (Figure 6). The HIFα subunits increase target gene transcription in hypoxic cells. However, HIF1α uniquely activates glycolytic enzyme genes, while HIF2α preferentially activates VEGF and cyclin D1. HIF2α promotes while HIF1α inhibits c-Myc transcriptional activity and cell cycle progression in RCC . HIF1α negatively regulates Wnt/β-catenin signaling, while HIF2α is required for β-catenin activation in RCC cells and for RCC proliferation . HIF1α/HIF2α imbalance in cancer cells might be important for RCC growth and metastasis.
The expression patterns of IL-6 and TNFα indicate that NF-κB signaling pathway is more active in MRCC than in NRCC. This result was later confirmed by the western blot findings that IκBα was more degraded in MRCC than in NRCC following the treatment with TNFα (Figure 7). Thus, NF-κB is not only critical in regulating RCC biology that pose challenge to conventional therapy , but also important in promoting ccRCC metastasis.