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Dynamic Change in p63 Protein Expression during Implantation of Urothelial Cancer Clusters.

Yoshida T, Okuyama H, Nakayama M, Endo H, Tomita Y, Nonomura N, Nishimura K, Inoue M - Neoplasia (2015)

Bottom Line: Here, we found that cancer cell clusters from the urine of patients with urothelial cancer retain the ability to survive, grow, and adhere.By using cell lines and primary cells collected from multiple patients, we demonstrate that △Np63α protein in cancer cell clusters was rapidly decreased through proteasomal degradation when clusters were attached to the matrix, leading to downregulation of E-cadherin and upregulation of N-cadherin.Our data provide the first evidence that clusters of urothelial cancer cells exhibit dynamic changes in △Np63α expression during attachment to the matrix, and decreased △Np63α protein plays a critical role in the interaction between cancer cell clusters and the urothelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Osaka Medical Center for Cancer and Cardiovascular Diseases; Department of Urology, Osaka University Graduate School of Medicine.

No MeSH data available.


Related in: MedlinePlus

Cancer cell clusters in the urine of patients with bladder cancer retain the ability to grow and adhere.(A) Hematoxylin and eosin staining of primary bladder urothelial tumors (upper row), phase contrast images (middle row), and Papanicolaou staining of sediments in urine derived from the same patients (lower row). HG, high grade. Scale bar, 100 μm. (B) Phase contrast images of cell clusters collected from the urine of a patient (BC289) with urothelial cancer and cultured in Matrigel growth factor reduced. The culture periods are indicated. Scale bar, 200 μm. (C) Immunostaining for Ki67 in cell clusters collected from the urine of a patient (BC287) with urothelial cancer. (D) Phase contrast images of cell clusters collected from the urine of a patient (BC297) with urothelial cancer and cultured in adhesive conditions. The culture periods are indicated. Scale bar, 200 μm. (E, F) Cell death assay by PI incorporation with single cells or cell clusters prepared from surgically resected urothelial tumors (BC298). Bright-field and fluorescent images of PI (E) and histograms of flow cytometry at day 2 (F). Red lines are for PI staining, and gray shades are for nonstained cells. (G, H) Bright-field and fluorescent images of PI (G) and histograms of flow cytometry at day 2 (H) with a bladder cancer cell line (5637). Scale bar, 200 μm.
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f0025: Cancer cell clusters in the urine of patients with bladder cancer retain the ability to grow and adhere.(A) Hematoxylin and eosin staining of primary bladder urothelial tumors (upper row), phase contrast images (middle row), and Papanicolaou staining of sediments in urine derived from the same patients (lower row). HG, high grade. Scale bar, 100 μm. (B) Phase contrast images of cell clusters collected from the urine of a patient (BC289) with urothelial cancer and cultured in Matrigel growth factor reduced. The culture periods are indicated. Scale bar, 200 μm. (C) Immunostaining for Ki67 in cell clusters collected from the urine of a patient (BC287) with urothelial cancer. (D) Phase contrast images of cell clusters collected from the urine of a patient (BC297) with urothelial cancer and cultured in adhesive conditions. The culture periods are indicated. Scale bar, 200 μm. (E, F) Cell death assay by PI incorporation with single cells or cell clusters prepared from surgically resected urothelial tumors (BC298). Bright-field and fluorescent images of PI (E) and histograms of flow cytometry at day 2 (F). Red lines are for PI staining, and gray shades are for nonstained cells. (G, H) Bright-field and fluorescent images of PI (G) and histograms of flow cytometry at day 2 (H) with a bladder cancer cell line (5637). Scale bar, 200 μm.

Mentions: We first examined the urine samples collected from patients with urothelial cancer to assess the status of cancer cells in the urine. We collected cancer cell clusters, known from clinical cytology to exist in patients’ urine, from patients’ urine (Figure 1A, Supplementary Figure 1). Some of the clusters (8 of 28 clusters from 5 patients, 28.6%) grew as spheroids in vitro with Ki67-positive proliferating cells (Figure 1, B and C). In addition, 5 of 12 clusters from 3 patients (41.7%) were able to attach to type I collagen–coated dishes (Figure 1D). Thus, the cancer cells also existed as clusters in the urine of patients with urothelial cancer and were able to grow and attach to the matrix. When cancer cells detach from the extracellular matrix, they usually die because of anoikis [33,34]. We previously reported that cancer cells from urothelial cancers, as well as other cancers from patient tumors, can be stably prepared and cultured by maintaining cell–cell contacts throughout the preparation and culture procedure using a CTOS method [27,28,31,35]. When the clusters of cancer cells prepared from surgically resected urothelial tumors were dissociated into single cells, the dead cells with PI-positive staining drastically increased after 24 hours in culture under floating conditions compared with intact clusters (Figure 1, E and F). The clusters formed spheroids with smooth surface after 24 hours (Figure 1E). The same phenomenon was observed in the bladder cancer cell line 5637 (Figure 1, G and H). These results suggest that urothelial cancer cells in the urine have a better chance of surviving as clusters than as single cells under floating conditions.


Dynamic Change in p63 Protein Expression during Implantation of Urothelial Cancer Clusters.

Yoshida T, Okuyama H, Nakayama M, Endo H, Tomita Y, Nonomura N, Nishimura K, Inoue M - Neoplasia (2015)

Cancer cell clusters in the urine of patients with bladder cancer retain the ability to grow and adhere.(A) Hematoxylin and eosin staining of primary bladder urothelial tumors (upper row), phase contrast images (middle row), and Papanicolaou staining of sediments in urine derived from the same patients (lower row). HG, high grade. Scale bar, 100 μm. (B) Phase contrast images of cell clusters collected from the urine of a patient (BC289) with urothelial cancer and cultured in Matrigel growth factor reduced. The culture periods are indicated. Scale bar, 200 μm. (C) Immunostaining for Ki67 in cell clusters collected from the urine of a patient (BC287) with urothelial cancer. (D) Phase contrast images of cell clusters collected from the urine of a patient (BC297) with urothelial cancer and cultured in adhesive conditions. The culture periods are indicated. Scale bar, 200 μm. (E, F) Cell death assay by PI incorporation with single cells or cell clusters prepared from surgically resected urothelial tumors (BC298). Bright-field and fluorescent images of PI (E) and histograms of flow cytometry at day 2 (F). Red lines are for PI staining, and gray shades are for nonstained cells. (G, H) Bright-field and fluorescent images of PI (G) and histograms of flow cytometry at day 2 (H) with a bladder cancer cell line (5637). Scale bar, 200 μm.
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Related In: Results  -  Collection

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f0025: Cancer cell clusters in the urine of patients with bladder cancer retain the ability to grow and adhere.(A) Hematoxylin and eosin staining of primary bladder urothelial tumors (upper row), phase contrast images (middle row), and Papanicolaou staining of sediments in urine derived from the same patients (lower row). HG, high grade. Scale bar, 100 μm. (B) Phase contrast images of cell clusters collected from the urine of a patient (BC289) with urothelial cancer and cultured in Matrigel growth factor reduced. The culture periods are indicated. Scale bar, 200 μm. (C) Immunostaining for Ki67 in cell clusters collected from the urine of a patient (BC287) with urothelial cancer. (D) Phase contrast images of cell clusters collected from the urine of a patient (BC297) with urothelial cancer and cultured in adhesive conditions. The culture periods are indicated. Scale bar, 200 μm. (E, F) Cell death assay by PI incorporation with single cells or cell clusters prepared from surgically resected urothelial tumors (BC298). Bright-field and fluorescent images of PI (E) and histograms of flow cytometry at day 2 (F). Red lines are for PI staining, and gray shades are for nonstained cells. (G, H) Bright-field and fluorescent images of PI (G) and histograms of flow cytometry at day 2 (H) with a bladder cancer cell line (5637). Scale bar, 200 μm.
Mentions: We first examined the urine samples collected from patients with urothelial cancer to assess the status of cancer cells in the urine. We collected cancer cell clusters, known from clinical cytology to exist in patients’ urine, from patients’ urine (Figure 1A, Supplementary Figure 1). Some of the clusters (8 of 28 clusters from 5 patients, 28.6%) grew as spheroids in vitro with Ki67-positive proliferating cells (Figure 1, B and C). In addition, 5 of 12 clusters from 3 patients (41.7%) were able to attach to type I collagen–coated dishes (Figure 1D). Thus, the cancer cells also existed as clusters in the urine of patients with urothelial cancer and were able to grow and attach to the matrix. When cancer cells detach from the extracellular matrix, they usually die because of anoikis [33,34]. We previously reported that cancer cells from urothelial cancers, as well as other cancers from patient tumors, can be stably prepared and cultured by maintaining cell–cell contacts throughout the preparation and culture procedure using a CTOS method [27,28,31,35]. When the clusters of cancer cells prepared from surgically resected urothelial tumors were dissociated into single cells, the dead cells with PI-positive staining drastically increased after 24 hours in culture under floating conditions compared with intact clusters (Figure 1, E and F). The clusters formed spheroids with smooth surface after 24 hours (Figure 1E). The same phenomenon was observed in the bladder cancer cell line 5637 (Figure 1, G and H). These results suggest that urothelial cancer cells in the urine have a better chance of surviving as clusters than as single cells under floating conditions.

Bottom Line: Here, we found that cancer cell clusters from the urine of patients with urothelial cancer retain the ability to survive, grow, and adhere.By using cell lines and primary cells collected from multiple patients, we demonstrate that △Np63α protein in cancer cell clusters was rapidly decreased through proteasomal degradation when clusters were attached to the matrix, leading to downregulation of E-cadherin and upregulation of N-cadherin.Our data provide the first evidence that clusters of urothelial cancer cells exhibit dynamic changes in △Np63α expression during attachment to the matrix, and decreased △Np63α protein plays a critical role in the interaction between cancer cell clusters and the urothelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Osaka Medical Center for Cancer and Cardiovascular Diseases; Department of Urology, Osaka University Graduate School of Medicine.

No MeSH data available.


Related in: MedlinePlus