We describe a novel syngeneic "pseudo-orthotopic" in vivo model of prostate cancer progression. Our model uses the dorsal skinfold chamber technique with fluorescence video microscopy and TRAMP-C2 tumor cells. The cells were transfected with a histone H2B-GFP fusion protein, permitting real-time measurement of tumor size, as well as mitotic and apoptotic indices. To generate a "pseudo-orthotopic" milieu, pieces of prostate tissue (10-15 mm2) from donor mice were implanted into the chambers of C57BL/6 mice. The prostate tissue grafted into the chambers retained its native vasculature, as determined by transplantation of prostate tissue from GFP transgenic mice. TRAMP-C2 prostate cancer tumor spheroids (25,000 cells) were implanted in the chamber. Without prostate tissue, TRAMP-C2 prostate tumors were poorly angiogenic, displayed low mitotic and apoptotic indices (0.7 x 10(-4)), and no significant tumor growth could be detected. TRAMP-C2 tumors growing on transplanted prostate tissue in the chamber on the other hand had mitotic indices in the order of 1.6 x 10(-4) and apoptotic indices in the order of 0.8 x 10(-4). Furthermore, tumors with stroma were highly angiogenic, and were fully vascularized within 7-10 days. During a 4-week observation period, the number of tumor cells increased by nearly 300%. We used the model to study the effects of surgical castration. The most profound response was a rapid vascular regression of the tumor vasculature. Castration also increased apoptotic indices within the tumor without significant changes in mitosis. This model may be utilized for the rapid analysis of new therapeutic candidates against prostate cancer.

BACKGROUND:Animal models are crucial to further our understanding of the mechanisms of (progressive) growth of prostatic cancer.MATERIALS AND METHODS:We have developed an intravital microscopic model based on the dorsal skinfold chamber technique in mice, allowing continuous measurements of growth and angiogenesis of small tumor spheroids. A histone H2B-GFP fusion protein has been introduced in our cell lines, allowing evaluation of mitotic and apoptotic indices. This system was used to evaluate the growth and angiogenesis of LnCAP, PC3 and DU145 tumor spheroids.RESULTS:LNCAP spheroids regressed rapidly, with complete tumor cell death within 7 days. PC3 spheroids regressed at a slower rate demonstrating a high apoptotic rate, though considerably lower than LNCAP. DU145 spheroids regressed the slowest, demonstrating considerably lower apoptotic rates and measurable mitotic rates.CONCLUSION:The inability to effectively the innate immune system by encapsulation prevents the survival of small human micro-tumor xenografts. Thus, the complex interactions between tumor cells and TAMs, which are pivotal in tumor biology, are extensively perturbed by "foreign" human tumor cells.

Pyrrole-imidazole polyamides bind DNA with affinities comparable to those of transcriptional regulatory proteins and inhibit the DNA binding activities of components of the transcription apparatus. If polyamides are to be useful for the regulation of gene expression in cell culture experiments, one pivotal issue is accessibility of specific sites in nuclear chromatin. We first determined the kinetics of uptake and subcellular distribution of polyamides in lymphoid and myeloid cells using fluorescent polyamide-bodipy conjugates and deconvolution microscopy. Then cells were incubated with a polyamide-chlorambucil conjugate, and the sites of specific DNA cleavage in the nuclear chromatin were assayed by ligation-mediated PCR. In addition, DNA microarray analysis revealed that two different polyamides generated distinct transcription profiles. Remarkably, the polyamides affected only a limited number of genes.

Brush borders which are localized at the apical face of enterocytes, are composed of thousands of stiff microvilli containing bundles of microfilaments made of actin. Their assembly occurs during terminal differentiation of the enterocytes when these cells migrate along the villus of the intestinal mucosa. The cell line HT 29 derived from a human colonic adenocarcinoma whose differentiation can be induced, can also be used as a model to study in culture the assembly of the intestinal brush border.

We studied the expression of villin, a microfilament-associated, actin-binding protein typical of brush-border microvilli, in a variety of human carcinomas by applying immunofluorescence microscopy to frozen sections and immunoblotting methods to tissue extracts using a rabbit antiserum and a monoclonal antibody specific for villin. All of the 24 primary and metastatic colorectal adenocarcinomas tested were uniformly and strongly positive for villin, with the immunocytochemical labeling concentrated at the luminal cell margin. In poorly differentiated tumor areas, rudimentary tubules were stained. All of the six tubular adenocarcinomas of the stomach studied as well as two adenocarcinomas of the gall bladder and a hepatocellular carcinoma were also villin-positive. Villin was detectable in 12 of 14 adenocarcinomas of the pancreas; in some of these cases, its distribution was heterogeneous. Among 21 renal cell carcinomas investigated, positivity for villin was seen in nine of 13 clear cell tumors (especially those of grade II), and in all four chromophilic cell tumors; however, all four chromophobe cell tumors studied were negative. Four of 11 endometrial but none of nine ovarian carcinomas were (uniformly or focally) villin positive. Of 18 adenocarcinomas of the lung studied, one was uniformly and four focally positive for villin, while the remainder were negative. All of the other epithelial tumors studied, including 12 adenocarcinomas of the breast and seven epithelial or biphasic pleural mesotheliomas, were villin negative. Our results show that the expression of villin in intestinal epithelial cells is consistently maintained in their corresponding carcinomas, even when the organized brush-border structure has been lost. The presence of villin in some endometrial and pulmonary adenocarcinomas--in contrast to its absence in the respective normal epithelia--suggests that this protein is newly expressed during hyperplasia, dysplasia, or carcinogenesis. Determining the presence or absence of villin and its immunocytochemical staining pattern in metastatic adenocarcinomas may be of some help in determining the type and site of the primary tumor.