Confirming cancer-stromal cell fusion.

<p>Representative cancer-stromal cell fusion events from the co-culture of RL-1 cells with hMSC-GFP cells are shown. <b>A</b>, A single fusion event at day 7 is shown in bright field, green fluorescence, and red fluorescence. The green and red fluorescence images are merged (merged fluorescence) to show the two nuclei of different fluorescence. <b>B</b>, Merged fluorescence images for 4 additional fusion events are shown, with events 1 and 2 recorded at day 7, and 3 and 4 at day 14. Arrows are used to indicate nuclei. All the images are shown at 200× magnification.</p

Tracking the fate of cancer-stromal hybrids.

<p>Representative morphologies of the hybrid cells during colony formation are shown. <b>A</b>, Two weeks into the culture, most of the hybrids contained two nuclei of similar fluorescence. No cell division was observed. <b>B</b>, Four weeks into the co-culture, hybrid cells adopted atypical morphology with multiple nuclei. No cell division was observed. <b>C</b>, Six weeks into the culture, the remaining hybrid cells became thin or narrow, with multiple nuclei in segments of the cell. <b>D</b>, Eight weeks into the culture, cell division became prevalent. The cell division was abnormal because it produced daughter cells in varied shapes and with reduced viability. For each view, a phase contrast image (top) and red fluorescence image (bottom) are shown. When necessary, arrows are used to indicate nuclei. All the images are shown at 200× magnification.</p

Morphologic changes in the derivative clones during colony formation.

<p>The morphology of a derivative colony was followed during the cloning process, growing from a single well of a 96-well plate to a single well of 24-well plate, to a single well of a 6-well plate, and to a 10 cm dish. For each view, a phase contrast image (top) and red fluorescence image (bottom) are shown. All the images are shown at 100× magnification.</p

Reduced colony formation in cancer-stromal fusion hybrids.

a<p>Wells containing a single cell 24 hours after the plating were enumerated.</p>b<p>Colonies from the wells containing a single cell were enumerated.</p>c<p>Data were from one colony formation assay.</p>d<p>Data were combined results from 4 repeated colony formation assays.</p

Characteristics of the spontaneous cancer-stromal cell fusion.

<p>RL-1 cells (<b>A</b>) and HPS-15 cells (<b>B</b>) are shown in separate culture. After 7 days of co-culture, spontaneous fusion could be seen (<b>C</b>). At higher magnification, the fused cell contained two nuclei, one fluorescently red and the other fluorescently pale (<b>D</b>). Cancer-stromal fusion was frequently seen in areas where RL-1 and HPS-15 formed close contact (<b>E</b>). In some cases, cells in the middle of a fusion could be seen (<b>F</b>). The two nuclei could be seen close to each other (<b>G</b>) or separated (<b>H</b>). For each view, a phase contrast image (top) and red fluorescence image (bottom) are shown. Arrows are used to indicate nuclei.</p

Time-dependence of cancer-stromal cell fusion.

<p>Co-cultures of RL-1 and HPS-15 cells were observed weekly for frequency of cell fusion. For each view, a phase contrast image (top) and red fluorescence image (bottom) are shown. Arrows are used to indicate cancer-stromal cell fusion events. All the images are shown at 40× magnification.</p

Genotypic and phenotypic changes in the derivative clones from cancer-stromal fusion.

<p>Genotypic and phenotypic parameters of the first 9 clones of the RL-1 and HPS-15 fusion hybrids were compared to those of the first 12 clones from control cloning. Compared to RL-1 clones, all the derivative clones lost Y chromosomes (<b>A</b> versus <b>B</b>). Detected by Western blotting, some of the derivative clones showed persistent AR expression even under androgen-deprivaton (<b>C</b> versus <b>D</b>). In these studies, cells were cultured for 48 hours in regular culture medium (C), androgen deprivation medium (−), and androgen deprivation medium containing 5 nM R1881 (+). The derivative clones were detected to express increased levels of PSA, even during androgen-deprivation (<b>E</b> versus <b>F</b>). When growth rate was assayed by MTT conversion, clones derived from cancer-stromal fusion displayed accelerated growth in androgen-independent fashion (<b>G</b> versus <b>H</b>). Data represent the mean of triplicate assays. For all the data points, standard deviation was less than 5% of the mean and is not shown.</p

Monoamine Oxidase A Inhibitor–Near-Infrared Dye Conjugate Reduces Prostate Tumor Growth

Development of anti-cancer agents with high tumor-targeting specificity and efficacy is critical for modern multidisciplinary cancer research. Monoamine oxidase A (MAOA), a mitochondria-bound enzyme, degrades monoamine neurotransmitters and dietary monoamines. Recent evidence suggests a correlation between increased MAOA expression and prostate cancer (PCa) progression with poor outcomes for patients. MAOA induces epithelial–mesenchymal transition (EMT) and augments hypoxic effects by producing excess reactive oxygen species. Thus, development of MAOA inhibitors which selectively target tumors becomes an important goal in cancer pharmacology. Here we describe the design, synthesis, and <i>in vitro</i> and <i>in vivo</i> evaluation of <b>NMI</b>, a conjugate that combines a near-infrared dye for tumor targeting with the moiety derived from the MAOA inhibitor clorgyline. <b>NMI</b> inhibits MAOA with low micromolar IC₅₀, suppresses PCa cell proliferation and colony formation, and reduces migration and invasion. In mouse PCa xenografts, <b>NMI</b> targets tumors with no detectable accumulation in normal tissues, providing effective reduction of the tumor burden. Analysis of tumor specimens shows reduction in Ki-67⁺ and CD31⁺ cells, suggesting a decrease of cell proliferation and angiogenesis and an increase in M30⁺ cells, indicating increased apoptosis. Gene expression profiles of tumors treated with <b>NMI</b> demonstrate reduced expression of oncogenes <i>FOS</i>, <i>JUN</i>, <i>NFKB</i>, and <i>MYC</i> and cell cycle regulators <i>CCND1</i>, <i>CCNE1</i>, and <i>CDK4/6</i>, along with increases in the levels of tumor suppressor gene <i>TP53</i>, cell cycle inhibitors <i>CDKN1A</i> and <i>CDKN2A</i>, and MAOA-downstream genes that promote EMT, tumor hypoxia, cancer cell migration, and invasion. These data suggest that <b>NMI</b> exerts its effect through tumor-targeted delivery of a MAOA-inactivating group, making <b>NMI</b> a valuable anti-tumor agent

Log-rank test of overall survival by race including all cases (N = 54, number of events = 47).

<p>Log-rank test of overall survival by race including all cases (N = 54, number of events  = 47).</p

Unmixed NRP-1, p-p65 and VEGF protein expression images from the mQDL of tissues from a Chinese patient who survived for 66 months (top panels) vs a patient who survived for 2 months (bottom panels).

<p>Unmixed NRP-1, p-p65 and VEGF protein expression images from the mQDL of tissues from a Chinese patient who survived for 66 months (top panels) vs a patient who survived for 2 months (bottom panels).</p