Keratin 6 expression correlates to areas of squamous differentiation in multiple independent isolates of As(+3)-induced bladder cancer

Abstract

This laboratory has shown that arsenite (As(+3)) exposure can cause the malignant transformation of the UROtsa human urothelial cell line. This single isolate formed subcutaneous tumors with a histology similar to human urothelial cell carcinoma. The tumors also displayed areas of squamous differentiation of the urothelial cells, an infrequent but known component of human bladder cancer. In the present study, five additional independent isolates of As(+3)-transformed urothelial cells were isolated and each was shown to produce subcutaneous urothelial cell tumors with a characteristic histology very similar to those described in the initial report. That there were underlying phenotypic differences in the six independent isolates was demonstrated when they were assessed for their ability to form tumors within the peritoneal cavity. It was shown that two isolates could form hundreds of small peritoneal tumor nodules, one isolate a moderate number of tumor nodules, and three isolates no or only one tumor nodule. The peritoneal tumors were also characterized for their degree of squamous differentiation of the urothelial cells and, while areas of squamous differentiation could be found, such differentiation was substantially reduced compared to subcutaneous tumors. Immunostaining for keratin 6 was tested as a potential marker for malignant urothelial cells that had undergone squamous differentiation. Keratin 6 was shown to consistently stain only cells having some evidence of squamous differentiation. Keratin 16 was shown to follow the staining pattern of keratin 6. The isolates and tumor heterotransplants were all examined for keratin 6, 16 and 17 mRNA and protein expression.

Figure 1. Phase contrast light microscopy of the six As⁺³ -transformed isolates demonstrating epithelial morphology in all isolates

Panel A is UTAs#1, B) is UTAs#2, C) is UTAs#3, D) is UTAs#4, E) is UTAs#5, F) is UTAs#6.

Figure 2. H&E Histology and Keratin 6 and 16 Immunostaining for As⁺³ -Transformed Tumor Heterotransplants Exhibiting Mild, Moderate and Prominent Squamous Differentiation

Panels A, B and C shows the histology (A), keratin 6 immunostaining (B), and keratin 16 immunostaining (C) of a subcutaneous tumor heterotransplant that exhibits a mild degree of squamous differentiation. Panels D, E, and F shows the histology (D), keratin 6 immunostaining (E) and keratin 16 immunostaining (F) of a subcutaneous tumor heterotransplant that exhibits a moderate degree of squamous differentiation. Panel G through L shows the histology (G, J), keratin 6 immunostaining (H, K) and keratin 16 immunostaining (I, L) of two subcutaneous tumor heterotransplants that both exhibit a prominent degree of squamous differentiation. The histology, keratin 6 immunostaining and keratin 16 immunostaining in each panel are serial sections of the respective tumor heterotransplant. Arrows illustrate representative areas of squamous differentiation while stars illustrate representative areas without squamous differentiation and a morphology similar to that seen in transitional cell carcinomas. Keratin 6 and 16 are strongly immunoreactive in areas of squamous differentiation, but negative or weak in areas of transitional cell morphology. Keratin pearls illustrated with a + were negative for the expression of keratin 6 and 16. All images represent a magnification of 200×.

Figure 3. Examples of the distribution of tumor nodules in abdomen after IP injection of As⁺³ -transformed UROtsa isolates in nude mouse

Panel A shows the injection site nodule on outside of peritoneum, B) Peritoneal (internal) tumor nodule, C) In situ view, peritoneal (arrows), greater omentum (*) and superficial pelvic cavity (+) tumors, D) In situ with intestines removed, deep pelvic cavity tumors (*), E) In situ, intestines removed, liver reflected upward. Liver (arrows) and splenic area (*) tumors, F) In bloc, liver, stomach, duodenum, spleen: Tumor at inferior liver surface (#) and splenic area (*), G) In situ, liver and intestines removed. Tumor deposits (*) on abdominal surface of diaphragm appear as whitish growths in contrast to normal (#) diaphragmatic surface, H) In situ, liver and intestines removed. Larger tumor deposits (+) and normal (#) diaphragmatic surface, I) In situ, reflection of large intestine cephalad, mesenteric tumors (*), J) In situ, intestinal and mesenteric tumors (*), K) In situ, intestines and liver removed. Tumor deposits in retroperitoneum and surrounding kidney (*).

Figure 4. Histology and Keratin 6 and 16 Immunoreactivity of Peritoneal Tumor Heterotransplants

Panel A: H&E histology of a peritoneal tumor formed by the UTAs#1 isolate. The tumor is composed of sheets of tumor cells with cells at the periphery having a morphology similar to transitional cell carcinoma (stars). Some cells in the center are larger with an enhanced eosinophilic or clear cytoplasm (arrows). Panel B: Keratin 6 immunostaining of a serial section to that shown in Panel A. The large cells with an enhanced eosinophilic or clear cytoplasm show strong staining for keratin 6 (arrows). The smaller cells with a transitional cell morphology show absent to moderate staining for keratin 6. Panel C: Keratin 16 immunostaining of a serial section to that shown in panel A and B. Keratin 16 staining is localized to the same areas as that of keratin 6, but there is less intensity and frequency of staining of keratin 16 compared to that of keratin 6. Panel D: H&E histology of a peritoneal tumor formed by the UTAs#3 isolate. The morphology is similar to that in Panel A except there are fewer areas of cells with an enhanced eosinophilic or clear cytoplasm (arrows). Panel E: Keratin 6 immunostaining for a serial section to that shown in Panel D. The large more eosinophilic cells or those with a clear cytoplasm show strong staining for keratin 6 (arrows) while the smaller cells with a transitional cell morphology show absent to moderate staining for keratin 6. Panel F: Keratin 16 immunostaining of a serial section to that shown in panel D and E. Keratin 16 staining is localized to the same areas as that of keratin 6, but again there is less intensity and frequency of staining of keratin 16 compared to that of keratin 6. The sections were chosen to illustrate areas that displayed intense keratin 6 immunoreactivity. Magnification is 200×.

Figure 5. Immunostaining of keratin 17 in subcutaneous and intraperitoneal tumor heterotransplants

A) Immunostaining of keratin 17 in subcutaneous tumor heterotransplants. The squamous differentiated areas in the center of the tumor nests are strongly positive for keratin 17(arrows), while the areas with urothelial or basal like morphology are negative or faintly positive (*). B) Immunostaining of keratin 17 in subcutaneous tumor heterotransplants. The peripheral basal like cells of tumor masses (arrow heads) are strongly positive for keratin 17 while the central areas are negative or very weakly positive. C) Immunostaining of keratin 17 in intraperitoneal tumor heterotransplants. The larger cells with more cytoplasm in the center of the tumor nest are positive for keratin 17 (arrows), whereas the peripheral areas with morphology resembling urothelial carcinoma (*) are negative or only faintly positive. D) Immunostaining of keratin 17 in intraperitoneal tumor heterotransplants. Areas with transitional or basal-like morphology are positive for keratin 17 (arrow heads) whereas most of the tumor composed of large tumor cells with abundant cytoplasm are negative.

Figure 6. Expression of Keratin 6, 16 and 17 mRNA and protein in UROtsa parent, As⁺³ -transformed isolates and subcutaneous tumor heterotransplants

A, C, E and G: Real time PCR analysis of keratin 6a, 6b, 16 and 17 in UROtsa parent and transformed isolates. B, D, F and H: Real time PCR analysis of keratin 6a, 6b, 16 and 17 in tumor heterotransplants. I: Western analysis of keratin 6, 16 and 17 in UROtsa parent and As⁺³ -transformed isolates. J: Western analysis of keratin 6, 16 and 17 in tumor heterotransplants. Statistical analysis consisted of ANOVA with Tukey post-hoc testing, performed by GraphPad PRISM 4. * denote a significant difference from UROtsa parental cells (p < 0.05). Real time PCR data is plotted as the mean+/−SEM of triplicate determinations.

Figure 7. Keratin immunofluorescent staining with DAPI counter stain in arsenite-transformed isolates

Keratin 6 staining is shown for (A) UROtsa parent; (B) As #1; (C) As #2; (D) As #3; (E) As #4; (F) As #5; (G) As #6. Keratin 6 positive staining was observed in all isolates, however, keratin 6 was found to organize into strongly stained intermediate filaments in only a subset of the isolates As #1 (B), As #3 (D), and As #4 (E). Strongly stained keratin 16 intermediate filaments are shown for the As #4 isolate (H) and strongly stained keratin 17 filaments are shown for the As #6 isolate (I). Controls included staining using the secondary-antibody only and were found to be negative for all isolates and for both secondary antibody conditions (not shown). Bar = 20 μm.