sepharose and Carcinoma--Renal-Cell

The cancer stem cell (CSC) theory depicts such cells as having the capacity to produce both identical CSCs (symmetrical division) and tumor-amplifying daughter cells (asymmetric division). CSCs are thought to reside in niches similar to those of normal stem cells as described for neural, intestinal, and epidermal tissue, are resistant to chemotherapy, and are responsible for tumor recurrence. We recently described the niche-like nature of mouse renal adenocarcinoma (RENCA) cells following encapsulation in agarose macrobeads. In this paper we tested the hypothesis that encapsulated RENCA colonies function as an in vitro model of a CSC niche and that the majority of cells would undergo chemotherapy-induced death, followed by tumor recurrence. After exposure to docetaxel (5 µg/ml), 50% of cells were lost one week post-treatment while only one or two cells remained in each colony by 6 weeks. Surviving cells expressed OCT4 and reformed tumors at 16 weeks post-treatment. Docetaxel-resistant cells also grew as monolayers in cell culture (16-17 weeks post-exposure) or as primary tumors following transplantation to Balb/c mice (6 of 10 mice) or NOD.CB17-Prkdc(scid)/J mice (9 of 9 mice; 10 weeks post-transplantation or 28 weeks post-exposure). These data support the hypothesis that a rare subpopulation of OCT4(+) cells are resistant to docetaxel and these cells are sufficient for tumor recurrence. The reported methodology can be used to obtain purified populations of tumor-initiating cells, to screen for anti-tumor-initiating cell agents, and to investigate the in vitro correlate of a CSC niche, especially as it relates to chemo-resistance and tumor recurrence.

Topics: Animals; Antineoplastic Agents; Carcinoma, Renal Cell; Cell Line, Tumor; Docetaxel; Drug Resistance, Neoplasm; Humans; Kidney Neoplasms; Mice, Inbred BALB C; Neoplastic Stem Cells; Octamer Transcription Factor-3; Paclitaxel; Sepharose; Taxoids

Cancer cells and their associated tumors have long been considered to exhibit unregulated proliferation or growth. However, a substantial body of evidence indicates that tumor growth is subject to both positive and negative regulatory controls. Here, we describe a novel property of tumor growth regulation that is neither species nor tumor-type specific. This property, functionally a type of feedback control, is triggered by the encapsulation of neoplastic cells in a growth-restricting hydrogel composed of an agarose matrix with a second coating of agarose to form 6- to 8-mm diameter macrobeads. In a mouse cell model of renal adenocarcinoma (RENCA cells), this process resulted in selection for a stem cell-like subpopulation which together with at least one other cell subpopulation drove colony formation in the macrobeads. Cells in these colonies produced diffusible substances that markedly inhibited in vitro and in vivo proliferation of epithelial-derived tumor cells outside the macrobeads. RENCA cells in monolayer culture that were exposed to RENCA macrobead-conditioned media exhibited cell-cycle accumulation in S phase due to activation of a G(2)/M checkpoint. At least 10 proteins with known tumor suppression functions were identified by analysis of RENCA macrobead-conditioned media, the properties of which offer opportunities to further dissect the molecular basis for tumor growth control. More generally, macrobead culture may permit the isolation of cancer stem cells and other cells of the stem cell niche, perhaps providing strategies to define more effective biologically based clinical approaches to treat neoplastic disease.

Topics: Animals; Carcinoma, Renal Cell; Cell Culture Techniques; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Coculture Techniques; HCT116 Cells; Humans; Kidney Neoplasms; Mice; Mice, Inbred BALB C; Sepharose; Species Specificity

The culture of tumor cell lines in three-dimensional scaffolds is considered to more closely replicate the in vivo tumor microenvironment than the standard method of two-dimensional cell culture. We hypothesized that our method of encapsulating and maintaining viable and functional pancreatic islets in agarose-agarose macrobeads (diameter 6-8 mm) might provide a novel method for the culture of tumor cell lines. In this report we describe and characterize tumor colonies that form within macrobeads seeded with mouse renal adenocarcinoma cells. Approximately 1% of seeded tumor cells survive in the macrobead and over several months form discrete elliptical colonies appearing as tumor cell niches with increasing metabolic activity in parallel to colony size. The tumor colonies demonstrate ongoing cell turnover as shown by BrdU incorporation and activated caspase-3 and TUNEL staining. Genes upregulated in the tumor colonies of the macrobead are likely adaptations to this novel environment, as well as an amplification of G(1)/S cell-cycle checkpoints. The data presented, including SCA-1 and Oct4 positivity and the upregulation of stem cell-like genes such as those associated with the Wnt pathway, support the notion that the macrobead selects for a subpopulation of cells with cancer stem cell or cancer progenitor properties.

Topics: Animals; Apoptosis; Carcinoma, Renal Cell; Cell Culture Techniques; Cell Growth Processes; Cell Line, Tumor; Coculture Techniques; Gene Expression; Humans; Kidney Neoplasms; Mice; Mice, Inbred BALB C; Neoplastic Stem Cells; Sepharose; Tumor Cells, Cultured

Use of the Hamburger-Salmon soft agar assay method for in vitro chemotherapy sensitivity testing of samples of renal cell carcinoma has been somewhat limited by a relatively low proliferation/evaluability rate for this tumor type (approximately 50%). The tritiated thymidine ([ 3H]-TdR) incorporation assay method of Tanigawa et al. (Cancer Res., 42: 2159, 1982) was compared to a standard optical colony counting assay technique. Fifty-seven different primary and five metastatic fresh samples of human renal cell carcinoma were studied. Evaluability rate by the [3H]-TdR assay was 90% (greater than or equal to 300 cpm control). In comparison, evaluability rate by optical colony counting was 43% for this group of tumors. [3H]-TdR incorporation increased with increasing tumor grade and increasing stage. Spindle cell tumors showed significantly higher cpm than other cell types. Twenty-three primary tumors were evaluable by both [3H]-TdR and colony counting methods. The correlation coefficient ("r") for regression lines for drug sensitivity data points (optical counting vs. [3H]-TdR) of these individual experiments ranged from 0.50 to 0.99 with a mean r +/- S.D. of 0.76 +/- 0.15. For all 260 paired drug response observations of 23 tumors exposed to different drugs, the correlation was very good with r = 0.71. Since the [3H]-TdR assay has an evaluability rate of approximately 90% for renal cell carcinoma, gives drug sensitivity information which correlates well with the colony counting endpoint and yields chemotherapy sensitivity information four days after sample accession, the [3H]-TdR assay may be a more useful method for study of human renal cell carcinoma in vitro chemotherapy sensitivity testing than standard colony counting techniques.

Topics: Antineoplastic Agents; Carcinoma, Renal Cell; Cell Count; Colony-Forming Units Assay; Humans; Kidney; Kidney Neoplasms; Neoplastic Stem Cells; Sepharose; Thymidine; Tritium; Tumor Stem Cell Assay