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

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

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

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

Figure 1. Anti-β2-M Ab sensitizes prostate cancer cells to radiation <i>in vivo</i>.

<p><b>A.</b> Radiation sensitivity of ARCaP_E and ARCaP_M prostate cancer cells by clongenic assay. (** p<0.01, *** p<0.001, Student’s t test). <b>B.</b> ARCaP_M cells are sensitized to radiation in the presence of anti-β2-M Ab using clongenic assay. (*p<0.03, *** p<0.008, ANOVA). <b>C.</b> Effect of anti-β2-M Ab (0.8 mg/kg) and radiation (15 Gy) on tumor growth in subcutaneous ARCaP_M xenograft nude mice model. (** p<0.01, Student’s t test).</p

Anti-β2-M Ab prevents tumor formation in spontaneous prostate cancer TRAMP mouse model.

<p><b>A.</b> Cell viability of TRAMP C1 and TRAMP C2 prostate cancer cells in response to anti-β2-M Ab. (***p<0.001, Student’s t test). <b>B.</b> Merged near infra-red and X-ray image of abdomen of TRAMP mice treated with control IgG and anti-β2-M Ab (n = 4). Representative parental mice used as additional control (C57BL/6 mice). The tumorigenecity of control IgG antibody group was 100% (n = 4) and the tumorigenecity of anti-β2-M Ab treated group was 25% (n = 4). <b>C.</b> H&E images of prostates of control IgG mice and anti-β2-M Ab treated mice (10X). <b>D.</b> Immune cell (T and B cells) numbers of wild type mice, control IgG mice and anti-β2-M Ab treated mice measured by flow cytometry. <b>E.</b> Body weights of TRAMP mice treated with IgG or anti-β2-M Ab.</p

Anti-β2-M Ab increases iron and decreases DNA repair enzymes in prostate cancer cells.

<p><b>A.</b> Iron staining in control and anti-β2-M Ab (5 µg/ml) treated cells using iron staining kit in ARCaP_M prostate cancer cell lines. <b>B.</b> Mitochondrial superoxide levels in response to anti-β2-M Ab treatment in a time and dose dependent manner in <b>i.</b> ARCaP_M, <b>ii.</b> ARCaP_E prostate cancer cell lines (***p<0.001, Student’s t test) and <b>iii.</b> p69 immortalized prostate epithelial cells using MitoSOX dye. <b>C.</b> Mitochondrial superoxide in ARCaP_M, KD_HFE1 and KD_HFE3 using MitoSOX dye (*p<0.05, Student’s t test). <b>D.</b> Expression of stress response proteins and DNA repair enzymes in C4-2B Neo control and β2-M knockdown cell lines. <b>i.</b>β2-M protein expression, <b>ii.</b> HSP27 and HSP70 protein expression and <b>iii.</b> NUDT1 and MPG protein expression. NUDT1 and MPG protein expression in response to anti- β2-M Ab treatment in LNCaP and C4-2 prostate cancer cells.</p