Bioconjugated quantum dots (QDs) provide a new class of biological labels for evaluating biomolecular signatures (biomarkers) on intact cells and tissue specimens. In particular, the use of multicolor QD probes in immunohistochemistry is considered one of the most important and clinically relevant applications. At present, however, clinical applications of QD-based immunohistochemistry have achieved only limited success. A major bottleneck is the lack of robust protocols to define the key parameters and steps. Here, we describe our recent experience, preliminary results and detailed protocols for QD-antibody conjugation, tissue specimen preparation, multicolor QD staining, image processing and biomarker quantification. The results demonstrate that bioconjugated QDs can be used for multiplexed profiling of molecular biomarkers, and ultimately for correlation with disease progression and response to therapy. In general, QD bioconjugation is completed within 1 day, and multiplexed molecular profiling takes 1-3 days depending on the number of biomarkers and QD probes used.

Purpose: Near-IR fluorescence imaging has great potential for noninvasive in vivo imaging of tumors. In this study, we show the preferential uptake and retention of two hepatamethine cyanine dyes, IR-783 and MHI-148, in tumor cells and tissues.Experimental Design: IR-783 and MHI-148 were investigated for their ability to accumulate in human cancer cells, tumor xenografts, and spontaneous mouse tumors in transgenic animals. Time-and concentration-dependent dye uptake and retention in normal and cancer cells and tissues were compared, and subcellular localization of the dyes and mechanisms of the dye uptake and retention in tumor cells were evaluated using organelle-specific tracking dyes and bromosulfophthalein, a competitive inhibitor of organic anion transporting peptides. These dyes were used to detect human cancer metastases in a mouse model and differentiate cancer cells from normal cells in blood.Results: These near-IR hepatamethine cyanine dyes were retained in cancer cells but not normal cells, in tumor xenografts, and in spontaneous tumors in transgenic mice. They can be used to detect cancer metastasis and cancer cells in blood with a high degree of sensitivity. The dyes were found to concentrate in the mitochondria and lysosomes of cancer cells, probably through organic anion transporting peptides, because the dye uptake and retention in cancer cells can be blocked completely by bromosulfophthalein. These dyes, when injected to mice, did not cause systemic toxicity.Conclusions: These two heptamethine cyanine dyes are promising imaging agents for human cancers and can be further exploited to improve cancer detection, prognosis, and treatment.

The epithelial-to-mesenchymal transition (EMT) is crucial for the migration and invasion of many epithelial tumors, including prostate cancer. Although it is known that ZEB1 overexpression promotes EMT primarily through down-regulation of E-cadherin in a variety of cancers, the soluble ligands responsible for the activation of ZEB1 have yet to be identified. In the present study, we investigated the role of insulin-like growth factor-I (IGF-I) in the regulation of ZEB1 during EMT associated with prostate tumor cell migration. We found that ZEB1 is expressed in highly aggressive prostate cancer cells and that its expression correlates directly with Gleason grade in human prostate tumors (P < 0.001). IGF-I upregulates ZEB1 expression in prostate cancer cells exhibiting an epithelial phenotype. In prostate cancer cells displaying a mesenchymal phenotype, ZEB1 inhibition reverses the suppression of E-cadherin protein and down-regulates the expression of the mesenchymal markers N-cadherin and fibronectin. Furthermore, ZEB1 blockade decreases migratory and invasive potential in ARCaP M compared with the control. These results identify ZEB1 as a key transcriptional regulator of EMT in prostate cancer and suggest that the aberrant expression of ZEB1 in prostate cancer cells occurs in part in response to IGF-I stimulation. [Cancer Res 2008;68(7):2479-88]

No abstract

Cancer is not a single cell disease. Aberrant cancer cells and their interactive microenvironment are needed for prostate cancer to progress to androgen independence and distant metastasis. It is highly plausible that newly evolved prostate cancer cell clones dominate cancer metastasis after cell-cell and cell-matrix interaction with the host microenvironment, rather than the selection or expansion of a preexisting prostate cancer cell clone(s). Based on this premise potential molecular targets in the microenvironment are especially emphasized. Further elucidation of the molecular mechanisms underlying tumor-stromal interaction may yield improved medical treatments for prostate cancer growth and metastasis.

Transformed fibroblasts coinoculated with epithelial cells accelerated the growth and shortened the latency period of human epithelial tumors in athymic mice. Addition of NbF-1 fibroblasts caused epithelial tumors to grow from five marginally tumorigenic or "nontumorigenic" (nontumorforming) human tumor cell lines or strains: PC-i (prostate), WH (bladder), , and cells derived from the ascites fluids of patients with metastatic renal pelvic or prostate cancers. Evidence for the human and epithelial nature of these experimental tumors was provided by histologic, immunohistochemical, Southern and dot-blot hybridization, and cytogenetic analyses. Transformed fibroblasts induced predominantly carcinosarcomas, whereas nontumorigenic fibroblasts (NIH 3T3) and lethally irradiated transformed fibroblasts induced exclusively carcinomas. The fibroblast-epithelial interaction appears to occur bidirectionally and does not result from cell fusion. Because coculture experiments in vitro did not demonstrate an increased cell proliferation, it appears that undefined host factors can influence tumor growth. This tumor model may be useful in drug-screening programs and in mechanistic studies of factors regulating human tumor growth and progression.Interactions between epithelium and mesenchyme mediate crucial aspects of normal development and are also believed to be important in neoplasia (1)(2)(3)(4). By employing tissuerecombination techniques, it has been demonstrated that the growth and differentiation of normal epithelium is regulated either inductively or permissively by neighboring mesenchymal components (5, 6). Studies of stromal-epithelial interaction in the prostate gland revealed that mesenchyme isolated from the fetal urogenital sinus (7, 8) but not from the prostate of newborns (9) or adults (10) can stimulate proliferation of well-differentiated adult epithelium. Mesenchymal mediation of sex steroid action on glandular epithelium has also been demonstrated. The growth of the prostate (7, 8), the regression of the mammary gland by androgen stimulation (11,12), and the growth (13) and the expression of progesterone receptors (14) by the mammary epithelium in response to estrogen have been shown to be mediated by the indirect action of sex steroids on the fibromuscular stroma.Stromal influences on epithelial neoplasia have also been documented in the salivary gland (15), the mammary gland (16), the urinary bladder (17), and the skin (18 Tumorigenicity Determinations. The athymic nude BALB/c mouse strain (20-25 g, Charles River Breeding Laboratories) was used in all experiments. Tumor volumes were calculated by the formula weight x length x height x 0.5236 (24). With the exception of the PC-3 cell line (25), our definition of "tumorigenicity" conformed to the published data. Dot Blot and Southern Hybridization. DNA was prepared from tissues and tumors according to the method described by Davis et al. (26), with slight modification to include treatment steps with proteinase K (0.2 mg/ml, Sigma) and RNase A (20 ,g/...

LNCaP lineage‐derived human prostate cancer cell lines C4‐2 and C4‐2B4 acquire androgen independence and osseous metastatic potential in vivo. Using C4‐2 and C4‐2B4 the goals of the current investigation were 1) to establish an ideal bone xenograft model for prostate cancer cells in intact athymic or SCID/bg mice using an intraosseous route of tumor cell administration and 2) to compare prostate cancer metastasis by administering cells either through intravenous (i.v.) or intracardiac administration in athymic or SCID/bg mice. Subsequent to tumor cell administration, prostate cancer growth in the skeleton was assessed by radiographic bone density, serum prostate‐specific antigen (PSA) levels, presence of hematogenous prostate cancer cells and histopathologic evaluation of tumor specimens in the lymph node and skeleton. Our results show that whereas LNCaP cells injected intracardially failed to develop metastasis, C4‐2 cells injected similarly had the highest metastatic capability in SCID/bg mice. Retroperitoneal and mediastinal lymph node metastases were noted in 3/7 animals, whereas 2/7 animals developed osteoblastic spine metastases. Intracardiac injection of C4‐2 in athymic hosts produced spinal metastases in 1/5 animals at 8–12 weeks post‐injection; PC‐3 injected intracardially also metastasized to the bone but yielded osteolytic responses. Intravenous injection of either LNCaP or C4‐2 failed to establish tumor colonies. Intrailiac injection of C4‐2 but not LNCaP nor C4‐2B4 cells in athymic mice established rapidly growing tumors in 4/8 animals at 2–7 weeks after inoculation. Intrafemoral injection of C4‐2 (9/16) and C4‐2B4 (5/18) but not LNCaP (0/13) cells resulted in the development of osteoblastic bone lesions in athymic mice (mean: 6 weeks, range: 3–12 weeks). In SCID/bg mice, intrafemoral injection of LNCaP (6/8), C4‐2 (8/8) and C4‐2B4 (8/8) cells formed PSA‐producing, osteoblastic tumors in the bone marrow space within 3–5 weeks after tumor cell inoculation. A stepwise increase of serum PSA was detected in all animals. Reverse transcription‐polymerase chain reaction (RT‐PCR) to detect hematogenously disseminated prostate cancer cells could not be correlated to either serum PSA level or histological evidence of tumor cells in the marrow space. We have thus established a PSA‐producing and osteoblastic human prostate cancer xenograft model in mice. Int. J. Cancer 77:887–894, 1998.© 1998 Wiley‐Liss, Inc.

ARCaP uniquely models the molecular basis of prostate cancer bone and adrenal metastases and epithelial to mesenchymal transition.