Germline Genetic Variation Modulates Tumor Progression and Metastasis in a Mouse Model of Neuroendocrine Prostate Carcinoma

<div><p>Neuroendocrine (NE) differentiation has gained increased attention as a prostate cancer (PC) prognostic marker. The aim of this study is to determine whether host germline genetic variation influences tumor progression and metastasis in C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of aggressive NEPC. TRAMP mice were crossed to the eight progenitor strains of the Collaborative Cross recombinant inbred panel to address this. Tumor growth and metastasis burden were quantified in heterozygous transgene positive F1 male mice at 30 weeks of age. Compared to wild-type C57BL/6J-Tg(TRAMP)824Ng/J males, TRAMP x CAST/EiJ, TRAMP x NOD/ShiLtJ and TRAMP x NZO/HlLtJ F1 males displayed significant increases in tumor growth. Conversely, TRAMP x WSB/EiJ and TRAMP x PWK/PhJ F1 males displayed significant reductions in tumor growth. Interestingly, despite reduced tumor burden, TRAMP x WSB/EiJ males had an increased nodal metastasis burden. Patterns of distant pulmonary metastasis tended to follow the same patterns as that of local dissemination in each of the strains. All tumors and metastases displayed positive staining for NE markers, synaptophysin, and FOXA2. These experiments conclusively demonstrate that the introduction of germline variation by breeding modulates tumor growth, local metastasis burden, and distant metastasis frequency in this model of NEPC. These strains will be useful as model systems to facilitate the identification of germline modifier genes that promote the development of aggressive forms of PC.</p></div

Genetic background influences prostate and seminal vesicle tumor burdens, and tumor associated mortality in TRAMP mice.

<p>(a) Kaplan-Meier survival curve for TRAMP F1 strains with a significantly increased tumor-associated mortality compared to wildtype C57BL/6J-Tg(TRAMP)824Ng/J (B6) mice. (b) Average age adjusted prostate tumor burden in TRAMP F1 strains. (c) Average age adjusted seminal vesicle tumor burden in TRAMP F1 strains. Dotted line at 0.6 g on <i>y</i>-axis represents the maximum weight of seminal vesicles recorded in transgene negative TRAMP F1 mice at 30 weeks of age. Bar graphs represent average weight of prostate tumor or seminal vesicles ± SEM. *<i>P</i><0.050 and **<i>P</i><0.001. Values in parentheses below <i>x</i>-axis represent the number of animals evaluated in each group.</p

Representative photomicrographs of metastatic lesions displaying NE phenotype in organs harvested from TRAMP F1 mice.

<p>Panels (a), (c) and (e) show H&E staining of the metastatic lesions in the lymph node, lungs and liver, respectively. Panels (b), (d) and (f) show strong positive IHC staining of metastatic PC cells with synaptophysin. Bar denotes 50 µm.</p

Representative photomicrographs of strain-specific differences in the expression of NE markers in prostate tumors from TRAMP F1 mice.

<p>Panels (a), (b) and (c) show H&E staining of the tumors; Panels (d), (e) and (f) show IHC staining of tumors with NE marker synaptophysin; Panels (g), (h) and (i) show IHC staining with FOXA1; Panels (j), (k) and (l) show IHC staining with FOXA2. Arrows point to normal prostate luminal cells, showing positive staining for FOXA1 and no staining for the NE markers, synaptophysin and FOXA2. Bar denotes 100 µm.</p

Effect of paternal genotype on metastatic efficiency of carcinoma cells in TRAMP mice.

<p>(<b>a</b>) Incidence of macroscopic metastases in lymph nodes, kidney and liver modulated by paternal genotype. Data not shown for TRAMP-PWK since macroscopic metastases were not observed in this strain. (<b>b</b>) Incidence of lung metastases modulated by paternal genotype. These incidence counts include the presence of isolated NE tumor cells, microscopic or macroscopic metastases in lung parenchyma. (<b>c</b>) Average age adjusted lymph node metastasis burden affected by paternal genotype. The total nodal metastasis burden for less enlarged lymph nodes (<0.1 g) was rounded to 0.1 g. Bar graphs represent average nodal burden ± SEM. *<i>P</i><0.050 and **<i>P</i><0.001 for comparisons <i>vs.</i> wildtype C57BL/6J-Tg(TRAMP)824Ng/J (B6). Data not shown for TRAMP-PWK since nodal metastases were not observed in this strain. Values in parentheses below <i>x</i>-axis represent the number of animals evaluated in each group.</p

P-Rex1 links mTOR signaling to Rac activation and cell migration

<p>Poster showing the most important results that are part of the publication "P-Rex1 Links Mammalian Target of Rapamycin Signaling to Rac Activation and Cell Migration" (See link below)</p

Morphology of reconstituted mammary glands of both strains into a background

<p><b>Copyright information:</b></p><p>Taken from "Association of estrogen receptor-α and progesterone receptor A expression with hormonal mammary carcinogenesis: role of the host microenvironment"</p><p>http://breast-cancer-research.com/content/9/2/R22</p><p>Breast Cancer Research 2007;9(2):R22-R22.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1868922.</p><p></p> Mammary cells were isolated from 2-month-old BALB/c or C57BL/6 mice and 5 μl of cell suspensions were transplanted into the right or left cleared mammary fat pads, respectively, of 21-day-old Swiss or syngeneic mice. At surgery pellets containing Eplus Pg or plus MPA were also implanted subcutneously into the backs of the animals. One month later, animals were killed and mammary glands excised and fixed. Microscopic examination revealed no changes between C57BL/6 or BALB/c repopulated Swiss /glands, whereas C57BL/6 cells transplanted in syngeneic glands were under-developed (100×, bar: 700 μm). A representative image of the WM or the H&E stained slides of the reconstituted mammary glands in E/Pg-treated mice is shown. Microsatellite studies confirmed the strain origin of the epithelial cells in the Swiss background. bp, base pairs; E, 17β-estradiol; H&E, hematoxylin and eosin; LCM, laser capture microdissection; MPA, medroxyprogesterone acetate; Pg, progesterone; WM, whole mount

PR expression in mammary glands treated with progestins for 2 months: Western blot studies

<p><b>Copyright information:</b></p><p>Taken from "Association of estrogen receptor-α and progesterone receptor A expression with hormonal mammary carcinogenesis: role of the host microenvironment"</p><p>http://breast-cancer-research.com/content/9/2/R22</p><p>Breast Cancer Research 2007;9(2):R22-R22.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1868922.</p><p></p> Mammary glands from the same groups shown in Figure 4 were processed to obtain nuclear extracts (as explained in Materials and methods) and used in Western blots. A representative experiment using C-19 PR Santa Cruz antibody is shown. Because different amounts of epithelial cells are present in the different extracts, the ratio of PR-A/PR-B in three different experiments is shown. *< 0.05, **< 0.01, ***< 0.001 treated versus control. PR, progesterone receptor

Effects of progestins in salivary gland morphology and in serum pituitary hormone levels

<p><b>Copyright information:</b></p><p>Taken from "Association of estrogen receptor-α and progesterone receptor A expression with hormonal mammary carcinogenesis: role of the host microenvironment"</p><p>http://breast-cancer-research.com/content/9/2/R22</p><p>Breast Cancer Research 2007;9(2):R22-R22.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1868922.</p><p></p> Salivary glands from control, MPA, or Pg-treated BALB/c or C57BL/6 mice. Pellets were implanted subcutaneously and the animals killed after 2 months. MPA increased the development of convoluted granular ducts (arrow). No differences were observed between both strains of mice (400×, bar: 120 μm). Serum PRL, Pg, GH, and IGF-I levels measured by radioimmunoassay in control BALB/c or C57BL/6 female mice and after 24 hours of MPA or Pg treatment. No differences were detected in C57BL/6 mice as compared with BALB/c mice. Furthermore, Pg and MPA treatment induced an increase in PRL levels in both strains (< 0.001, two-way ANOVA). The increase in Pg levels after progesterone administration was also similar in both strains (< 0.001, two-way ANOVA). Gray bars: BALB/c; black bars: C57BL/6. ANOVA, analysis of variance; GH, growth hormone; IGF, insulin-like growth factor; MPA, medroxyprogesterone acetate; Pg, progesterone; PR, progesterone receptor; PRL, prolactin

Lymph node (LN) cells activated by an anti-CD3 antibody induce BMMSC apoptosis <i>in vitro</i>.

<p>(A) Mouse BMMSCs co-cultured with LN cells indicated that anti-CD3 antibody activated LN cells were capable of inducing BMMSC death as shown a blank well without BMMSC staining (blue). When co-cultured BMMSCs (MSC) and LN cells were separated by a transwell culture system, anti-CD3 antibody treated LN cells failed to induce BMMSC death. (B) It is known that immunocompromised mice have no T lymphocytes. Thus, LN cells derived from immunocompromised failed to induce BMMSC death following anti-CD3 antibody activation in the co-culture system. (C) TUNEL staining showed that BMMSC death caused by anti-CD3 antibody-activated LN cell is through an apoptotic pathway. (D) Condition medium (CM) derived from naïve LN cells and anti-CD3 antibody activated LN cells were not able to induce cell death of BMMSCs. (E) Neutralizing anti-TNF-α and IFN-γ antibodies were not able to inhibit BMMSC death induced by anti-CD3 antibody-activated LN cells. (F) Neutralizing Fas ligand antibodies and brefeldin A, but not concanamycin A, were capable of blocking BMMSC death induced by anti-CD3 antibody-activated LN cells. (G) Western blot analysis showed that mouse and human BMMSCs (mMSC and hMSC) express Fas. (H) Fas antibody can induce significant reduction in number of living BMMSCs in culture. (I) Anti-CD3 antibody-activated LN cells were not able to induce cell death of BMMSCs derived from <i>CD95</i>-deficient mice (<i>lpr</i>). (n = 5; [<i>P</i><0.05 and [[[<i>P</i><0.005).</p