HDL Levels Affect Tumor Development in Vivo To initially examine the effect of apoA1 on tumor progression and metastasis, we used B16F10L melanoma tumor cells and syngeneic animal hosts either lacking apoA1 (A1KO) and having low HDL levels (19.1 ± 1.7 mg/dl HDL cholesterol), wild type (WT) C57BL/6J mice (52.0 ± 4.5 mg/dl HDL cholesterol), or mice overexpressing the human apoA1 transgene (A1Tg+/−, 110.6 ± 7.5 mg/dl HDL cholesterol) in which murine apoA1 is markedly suppressed (34). Following subcutaneous inoculation of tumor cells bilaterally on the dorsal surface of mice, tumor growth was monitored and observed to be >10-fold reduced in A1Tg+/− mice compared with A1KO (Fig. 1A, p < 0.05). In parallel studies, metastasis growth was monitored in real time by in vivo bioluminescent imaging of the ventral surface of animals inoculated instead with a B16F10L variant stably expressing Renilla luciferase. Similarly, A1Tg+/− mice exhibited a >10-fold reduction in metastasis development compared with A1KO mice (Fig. 1B, p < 0.05). To determine whether the anti-neoplastic effect of apoA1 was unique to malignant melanoma tumor growth, parallel experiments were performed inoculating Lewis lung tumor cells, another syngeneic tumor model in C57BL/6J mice. Growth of Lewis lung tumors was also significantly (p < 0.05) inhibited in A1Tg+/− mice, and with even further tumor growth inhibition in animals homozygous for the human apoA1 gene (A1Tg+/+, Fig. 1, C and D, p < 0.05). These results thus revealed dose-dependent reduction in tumor growth with increasing apoA1 levels, with A1KO mice showing the greatest rates of tumor growth, and A1Tg+/+ mice, which express human apoA1 at ∼1.5-fold higher levels relative to A1Tg+/− mice, showing minimal tumor growth (Fig. 1, C and D). Because tumor growth and metastases in A1Tg+/+ animals was so markedly inhibited and as a practical matter, in further studies outlined below, we often employed A1Tg+/− mice in experiments designed to probe mechanisms of apoA1 anti-tumor activity. A1Tg+/− mice provided sufficient amounts of tumor tissues to be harvested to obtain enough material to isolate specific cells or to determine mRNA, protein, and enzymatic activity levels. View larger version: Download as PowerPoint Slide FIGURE 1. Enhanced tumor growth and metastasis in apoA1 null (A1KO) mice. Syngeneic C57BL/6J mice of indicated genotypes were inoculated with B16F10L melanoma (A), B16F10L-luciferase melanoma (B), or Lewis Lung (C and D) tumor cells. Tumor volume at the site of inoculation was monitored by caliper measurements (A, C, and D). D reflects Lewis lung tumor volume on day 48. Metastasis was measured by in vivo bioluminescence (B). Data points are mean values ± S.E.

Pharmacological Delivery of ApoA1 Prevents Tumor Growth and Metastasis The above results suggested pharmacological administration of apoA1 may provide therapeutic benefit as an anti-tumor agent. To test this hypothesis, the B16F10L melanoma model was examined due to its rapid and widely metastatic disease course. A regimen of subcutaneous injection of human apoA1 on alternating days was empirically developed in A1KO animals resulting in maintenance of plasma apoA1 levels within the normal physiological range observed in humans (trough 79 ± 7 versus peak 151 ± 9 mg/dl; data not shown). In initial studies, apoA1 (15 mg/animal) was administered prior to tumor inoculation to optimize potential observed effects, and in later studies (see below), the impact of apoA1 therapy on pre-existing (palpable) tumors and metastasis was tested. Remarkably, apoA1 injections provided extraordinary protection from tumor growth and metastasis compared with the vehicle control (normal saline) (Fig. 2). Tumor growth was readily detected at the site of tumor cell inoculation in the A1KO control group within several days, whereas the lack of luminescence in the apoA1-treated group indicated a complete failure of tumor growth and development. Quantitative analyses of the bioluminescence measurements indicated apoA1 treatment inhibited both tumor growth and metastasis by over 100-fold (Fig. 2, B and C, p < 0.05 each). Further illustration of the dramatic reduction in tumor growth afforded by apoA1 injection is illustrated in Fig. 2D, in which a representative animal (with fur shaved for bioluminescence analyses) from each treatment group is shown on day 21 post-tumor inoculation. Survival analyses similarly showed dramatic protection afforded by apoA1 injections, with 100% survival in the apoA1-treated animals throughout the duration of the study (terminated on day 35). This contrasted with a 50% reduction in survival in the normal saline-treated group by day 21, and with all animals in this arm dead by day 29 (Fig. 2E). View larger version: Download as PowerPoint Slide FIGURE 2. ApoA1 therapy confers resistance to melanoma development and improves survival in A1KO mice. Human apoA1 (15 mg per animal) or normal saline were administered in A1KO mice starting 3 weeks prior to tumor inoculation (B16F10L-luciferase melanoma) and continued for the duration of the experiment. Tumor progression was monitored by live (bioluminescent) imaging. A, representative image of the dorsal view taken on day 16 post-tumor inoculation. Tumor burden (B) reflects the sum of luminescence from both dorsal and ventral views, although only the ventral view was used to quantify metastasis (C). D, representative animals from each treatment arm are shown 21 days post-tumor inoculation. Survival plot is shown in E. Data points are mean ± S.E. To examine the impact of apoA1 on existing tumor and metastasis, A1KO animals were inoculated with B16F10L-luciferase tumor cells, and once both tumors were palpable and metastasis was detected (day 6), subcutaneous apoA1 injections versus normal saline treatments were initiated. Within 1 week, a significant 2-fold reduction in peak tumor and metastasis burden in the apoA1-treated group was observed (Fig. 3, A and B (inset); p < 0.05 for each). With longer duration of therapy, the apoA1-treated animals showed nominal further tumor growth and metastasis, whereas the normal saline-injected group succumbed to progressive melanoma tumor development (Fig. 3, A and B). These data indicate that apoA1 not only inhibits or delays the development of tumor progression (Fig. 2) but, more importantly, apoA1 therapy can shrink and retard further development of both established tumor and metastasis (Fig. 3, A and B). View larger version: Download as PowerPoint Slide FIGURE 3. ApoA1 but not apoA2 therapy promotes regression of established melanoma tumors and metastasis. A1KO mice were inoculated with B16F10L-luciferase melanoma. Six (A and B) or seven (C and D) days post-tumor inoculation when tumor was palpable and in vivo imaging showed metastasis, injection of human apoA1 (20 mg per animal); A–D, apoA2 (20 mg per animal); C and D, or normal saline (A–D) was initiated and continued every other day for the duration of the experiment. Tumor burden (sum of luminescence from dorsal and ventral sides) is shown in A and C, and metastasis (luminescence from ventral side) is shown in B and D. Insets (A and B) show data for the apoA1 treatment arm on an expanded scale to better illustrate tumor progression/regression before and after apoA1 treatment. Data points are mean ± S.E. The specificity of the anti-neoplastic biological activity of apoA1 was next examined by comparing the effect of subcutaneous injections with either apoA1 or the second most abundant HDL-associated protein, apolipoprotein A2 (apoA2). A1KO mice were again first inoculated with B16F10L-luciferase tumor cells, and once tumors were both palpable and metastasis observed, subcutaneous injections of equivalent amounts (by mass) of either apoA1 or apoA2 were initiated. Remarkably, although apoA1 again promoted tumor and metastasis regression, apoA2 showed no beneficial effect on either tumor growth or metastasis (Fig. 3, C and D). Interestingly, apoA2-treated animals showed a tendency toward enhanced tumor burden and metastasis relative to the normal saline arm, but the differences did not reach statistical significance (Fig. 3C, p = 0.36, day 19; Fig. 3D, p = 0.15, day 19).

Anti-tumor Effects of ApoA1 Are Indirect and Not Associated with Altered Antigen Presentation or Systemic Lysophosphatidic Acid Levels Efforts to reveal the mechanism(s) through which apoA1 mediates its anti-neoplastic activity initially focused on studies examining whether the lipoprotein exhibits a direct effect on cancer cells, such as reductions in proliferation or apoptosis rates. However, in vitro treatment of B16F10L tumor cells with physiological levels of either apoA1 or reconstituted nascent HDL demonstrated no significant alterations in cellular proliferation rate, various cell cycle parameters, or apoptosis rate, indicating there is no major direct inhibitory effect on the tumor cells with either apoA1 or the lipid-associated lipoprotein (data not shown). Recent studies suggest lipid accumulation in the form of triglycerides within dendritic cells (DCs) can significantly inhibit their ability to present tumor antigen and activate T cells (35). We therefore sought to determine whether DCs in A1KO mice might be functionally defective leading to accelerated tumor growth and whether, alternatively, DCs from A1Tg mice showed enhanced capacity to stimulate allogeneic T cells. CD11c+ DCs were isolated from spleens of naive or B16F10L-bearing A1KO and A1Tg mice, and their capacity to promote allogeneic T cell proliferation was determined in mixed lymphocyte proliferation assays. DCs recovered from tumor-bearing A1KO and A1Tg+/+ mice both stimulated allogeneic T cells to an equivalent extent (p = 0.99 for comparison between CD11c+ cells from A1KO versus A1Tg mice), each mediating a 3-fold increase in T cell proliferation as compared with DCs isolated from naive A1KO and A1Tg+/+ mice (Fig. 4). The increase in T cell proliferation observed in the tumor-bearing mice is consistent with tumor-induced mobilization of DCs (36). However, the lack of demonstrable differences between DCs recovered from A1KO and A1Tg mice argues against an in vivo role for apoA1 in modulating tumor antigen presentation by DCs and subsequent T cell activation. View larger version: Download as PowerPoint Slide FIGURE 4. ApoA1 anti-tumor activity is independent of DC function. Allogeneic T cell proliferation induced by splenic DCs. Immunopurified CD11c+ DCs were isolated from spleens of naive and tumor-bearing A1KO or A1Tg+/+ animals 14 days post-tumor (B16F10L-luciferase) inoculation. Increasing numbers of irradiated pooled DCs were mixed with a fixed number of T cells (CD3+) isolated from naive wild type BALB/c splenocytes, and T cell proliferation was assessed by [3H]thymidine uptake as described under “Experimental Procedures.” Studies by Farias-Eisner and co-workers (29) recently reported that injection of short anti-inflammatory amphipathic helical peptides, initially developed as apoA1 mimetics for cardiovascular disease, can inhibit ovarian cancer, presumably through binding of the pro-inflammatory lipid, LPA. We therefore quantified plasma levels of multiple distinct molecular species of lysophosphatidic acid in tumor-bearing A1KO, WT, and apoA1Tg mice using stable isotope dilution LC-MS/MS analyses. In contrast to results reported with the amphipathic peptides, no significant reductions in any LPA species were noted (Table 1). The above authors also demonstrated a significantly higher LPA binding capacity of the amphipathic peptides as compared with full-length apoA1 protein and a correlation between plasma LPA levels and tumor progression (29). The absence of any correlation between plasma levels of LPA molecular species and tumor growth in our present studies, along with the poor LPA binding activity previously noted with full-length apoA1 (29), indicates a distinct mechanism of anti-tumor activity is present with full-length apoA1 protein (this study) as compared with the short amphipathic peptides previously reported. View this table: TABLE 1 Plasma concentration of lysophosphatidic acid species (nanomolar) in tumor-bearing mice The indicated numbers of C57BL/6J mice were inoculated with 105 B16F10L-luciferase melanoma tumor cells per flank. Plasma was recovered from tumor-bearing animals 17 days post-tumor inoculation. Individual molecular species of LPA were quantified by LC-MS/MS as described under “Experimental Procedures.” Data are presented as mean ± S.E. p values were calculated by two-tailed Student's t test. Note that no significant difference between any of the groups for any of the LPA molecular species or the total of all LPA species was observed.

Anti-tumor Effects of ApoA1 Require an Immunologically Intact Host In the absence of any apparent direct effect on tumor cells, changes in antigen presentation and T cell proliferative responses in vitro, or involvement of LPA sequestration as a potential mechanism, we investigated whether the anti-tumor effect of apoA1 was mediated via other changes in the host immune response to tumor. To test this hypothesis, we examined whether apoA1 was inhibitory to melanoma progression in mice deficient in various aspects of the immune system. B16F10L melanoma cells were inoculated into the severely immunodeficient NSG mice, which lack mature lymphocytes (B and T cells), natural killer cells, and fully functional DCs (37). ApoA1 injections resulted in an approximate 5-fold reduction in tumor growth in the NSG mice (Fig. 5A). Because tumor development was still significantly repressed in response to apoA1 therapy in these immunocompromised mice, these data suggest that an appreciable portion of the anti-neoplastic activity observed with apoA1 is independent of adaptive immunity, as the scid defect within NSG mice effectively eliminates adaptive immunity. In contrast, apoA1-induced B16F10L tumor repression in the fully immunocompetent A1KO mice (Fig. 5B) was nearly complete compared with that observed in the NSG mice (Fig. 5A), indicating that elements of both innate and adaptive immunity are required for full anti-neoplastic activity by apoA1. To examine if apoA1 therapy can be extended to human tumor xenografts, nude mice, which lack T cells and show both a partial defect in B cell development and a lack of cell-mediated immunity, were inoculated with human melanoma (A375) cells, followed by either apoA1 or normal saline injections. Interestingly, tumor development was significantly retarded by nearly 2-fold in the apoA1-treated animals (p = 0.02); however, tumor growth still proceeded within these apoA1-treated nude mice (Fig. 5C). These results indicate that although a defect in T cell immunity (Fig. 5C) partially attenuates the anti-neoplastic activity of apoA1 (Fig. 5B), a significant portion of the biological effect of apoA1 is mediated by T cell-independent mechanisms. Importantly, the results in Fig. 5C also suggest apoA1 anti-tumor activity extends to human tumors. We conclude that elements of both innate and adaptive immunity are required for full anti-neoplastic activity by apoA1. Nude and NSG mice have normal levels of murine apoA1. Inhibition of tumor growth in these mice is supportive of the relevance of the apoA1 effect under normal physiological conditions where apoA1 levels are intact. View larger version: Download as PowerPoint Slide FIGURE 5. ApoA1 anti-neoplastic activity involves both innate and adaptive immunity. Human apoA1 injections (20 mg, s.c., every other day) were initiated on day 1 in NSG mice (A), A1KO mice (B), and Nude mice (C). Mean tumor volumes in mice inoculated with B16F10L-luciferase melanoma cells (A and B, day 21) or human melanoma A375 cells (C, day 20) are shown. Horizontal bars represent median tumor volumes of the data series.

ApoA1 Inhibits MDSC Expansion and Recruitment into the Tumor Environment We next explored a potential role for MDSCs as a possible target for apoA1 actions. MDSCs play a pivotal role in tumor-induced immune suppression and constitute a major component of the leukocyte infiltrate in the tumor (38). MDSCs are a heterogeneous population of immature myeloid cells that have the properties of macrophages, granulocytes, and DCs and whose numbers normally expand in response to tumor-driven factors, including prostaglandins, CXCR2, VEGF, and GM-CSF (38, 39). In initial studies we examined the impact of reconstituted nascent HDL on cultured bone marrow-derived macrophages. Of note, in the presence of HDL, expression levels of Ptgs1 (COX-1) and Cxcl2, the ligand for CXCR2, were significantly (p < 0.01 each) reduced (supplemental Table 1). The in vivo relevance of an apoA1 effect on MDSC was therefore next examined by testing whether A1Tg mice might accumulate fewer peripheral (splenic) MDSCs in response to tumor challenge. Flow cytometry analyses of splenic cells harvested 14 days after either vehicle (i.e. naive) or tumor cell inoculation revealed that in naive tumor-free mice, CD11b+GR-1+ (MDSC) cells showed only small numbers in the spleen (Fig. 6A). Myeloid differentiation antigen GR-1 consists of two epitopes, LY-6G and LY-6C, and MDSCs consist of two major subsets, the granulocytic CD11b+LY-6GhighLY-6Clow (CD11b+GR-1high) and the monocytic CD11b+LY-6GlowLY-6Chigh (CD11b+GR-1low) populations. Further flow cytometry analyses revealed a significant (p = 0.03) 2-fold tumor-induced increase in the percentage of splenic MDSCs recovered from A1KO mice, whereas no significant increase was observed in splenic MDSCs recovered from A1Tg+/+ mice (Fig. 6A). These data suggest expansion of splenic MDSC in response to the B16F10L tumor is attenuated in A1Tg+/+ mice. Confirmation of this was observed in a subsequent experiment in which tumor-bearing spleens from A1KO and A1Tg+/− mice were examined 12 days post-tumor cell inoculation. Decreased expansion of CD11b+GR-1+ MDSCs was noted in the A1Tg+/− versus A1KO tumor-bearing mice (3.9% versus 5.9%, respectively; p = 0.02, Fig. 6B). Of note, mature macrophages (indicated by F4/80+ staining) were also significantly decreased in the spleens of the A1Tg+/− versus A1KO tumor-bearing mice (9.1% versus 11.8%; p = 0.01, Fig. 6B). View larger version: Download as PowerPoint Slide FIGURE 6. ApoA1 therapy inhibits accumulation of MDSCs in tumor bed. A and B, flow cytometry was performed on splenocytes from indicated genotypes inoculated with normal saline (naive) or B16F10L-luciferase melanoma cells (two sites, 1 × 105 cells per site) (A) or inoculated with B16F10L-luciferase melanoma cells at four separate sites (B, 1 × 105 cells per site) and sacrificed on day 14 (A) or day 12 (B) post-tumor inoculation. Data points are mean ± S.E. C, representative day 7, B16F10L-luciferase tumor staining from A1KO and A1Tg+/− mice for GR1+ cells. Main image is at ×10 and the inset is at ×40 magnification with scale bars representing 20 μm. D, subcutaneous day 15 B16F10L-luciferase tumors were excised from A1KO tumor-bearing mice treated with apoA1 therapy (20 mg/day/animal on days 10–14 post-tumor inoculation; 105 tumor cells/site; six sites per animal) and pooled (n = number of animals/treatment arm). Tumors were digested, and single cell suspensions were surface-stained for MDSCs (CD11b+GR1+) and processed by flow cytometry as described under “Experimental Procedures.” Immune cells are expressed as frequency of live CD45.2+ cells (tumor-infiltrating leukocytes; TILs). Current tumor immunobiology paradigms imply that MDSCs in tumor-bearing animals contribute to tumor progression, increased angiogenesis, and decreased immune surveillance, with MDSC levels being elevated not only in the circulation and peripheral immune organs but also in and around the tumor itself (38, 40). Consistent with our observation of decreased CD11b+GR-1+ cells in the spleens of tumor-bearing A1Tg mice, examination of B16F10L primary tumor tissue for the presence of CD11b+GR-1+ cells revealed that these cells were easily detected in primary tumors in A1KO mice but were significantly less abundant in A1Tg+/− mice (Fig. 6C shows a representative example). Importantly, apoA1 therapy (days 10–14 post-tumor inoculation) in A1KO mice reduced (by 50% compared with control saline-injected) the accumulation of CD11b+GR-1+ in primary tumors (Fig. 6D). Together, the above results indicate an inhibitory role for apoA1 in MDSC expansion and recruitment to the tumor microenvironment.

Tumor-associated Angiogenesis and Tumor Invasion Are Inhibited by ApoA1 Tumor neoangiogenesis is required for tumor growth and metastasis. The dramatic inhibitory effect of apoA1 on tumor growth and metastasis suggested another potential mechanism through which apoA1 may exert its anti-neoplastic effect(s) may be via inhibition of angiogenesis. Quantitative analyses of blood vessels directly feeding into day 7 primary mouse melanoma tumors revealed a significant (p < 0.005) >2-fold decrease in the number of vessels in tumors from A1Tg+/+ mice compared with A1KO hosts (Fig. 7A), consistent with apoA1 having an inhibitory effect on neoangiogenesis in the tumor bed. More detailed comparisons in A1Tg+/+ versus A1KO mice revealed marked reduction in multiple key properties of the vascular network feeding the tumor within the A1Tg+/+ mice, including reduction in vessel area, vessel length density, and size of vessels (p < 0.005 for each; Fig. 7, C–E). Thus, primary tumors from animals with the same initial tumor burden exhibited decreased angiogenesis in the presence of apoA1. View larger version: Download as PowerPoint Slide FIGURE 7. Reduced angiogenesis in primary tumors from A1Tg mice. Angiogenesis was assessed in B16F10L-luciferase melanoma bearing mice 7 days post-tumor inoculation. A and B, number of vessels feeding directly into the primary tumor was counted under a microscope as described under “Experimental Procedures.” Images of tumor were captured at ×12.5 magnification (B, left). The region of interest (B, middle; white) representing the tumor mass defined the perimeter of the tumor. The output was a series of color generation maps (colored vessels on black background) in which the largest diameter vessels were defined as G 1 (red), with each subsequent smaller generation represented as G 2 –G 6 (B, right). These images were used to quantitate total vessel area (C), vessel length density (D), and size of vessels (E), as described under “Experimental Procedures.” F, day 7 B16F10L-luciferase subcutaneous tumors were homogenized for RNA or protein as described under “Experimental Procedures.” F, left panel, TaqMan assays for murine Vegfa were normalized to B2m and expressed as relative quantification (RQ). Bar graphs represent mean of the data series. F, right panel, VEGF dimer protein as detected by Western blot and normalized to β-actin. Horizontal bars represent median of the data series. n = number of tumor inoculation sites in A–E or the number of animals in F. Angiogenesis can be vascular endothelial growth factor (VEGF-A)-dependent or -independent (41, 42). Examination of Vegfa mRNA expression and VEGF protein levels from tumor tissue recovered 7 days post-cancer cell inoculation in A1Tg animals exhibited a modest increase relative to that observed in A1KO animals (Fig. 7F). Thus, the reduction in angiogenesis observed within the A1Tg animals does not appear to be related to VEGF. This result contrasts with the recent finding that in an ovarian cancer model short amphipathic peptide mimetics of apoA1 directly reduced the viability of tumor cells in tissue culture and inhibited production of VEGF (28).

ApoA1 Inhibits MMP-9 Matrix metalloproteinases are zinc-dependent extracellular matrix-degrading enzymes critical for tumor angiogenesis, invasion, and metastasis in many cancers. Higher levels of active MMP-9 have been observed in more invasive and metastatic melanoma tumors (43). Expression of latent MMP-9 is regulated at the level of transcription, whereas its enzymatic activity is controlled by specific proteolytic cleavage of pro-MMP-9 (43). We therefore investigated whether apoA1 or reconstituted nascent HDL could lead to decreased levels of MMP-9 that would negatively affect cell invasion through a basement membrane. Initially, we tested this idea directly on B16F10L tumor cells in vitro using a cell invasion assay. Exposure of cells to plasma levels of either HDL or apoA1 resulted in modest (∼10%) but statistically significant impairment in tumor cell invasion across the basement membrane (data not shown). Given this small direct effect of the lipoprotein on B16F10L cell invasion in vitro, we next examined if this effect might be amplified in vivo and result as a consequence of decreased MMP-9 levels in tumors. Although no significant differences in Mmp9 mRNA level from day 7 melanoma tumors harvested from A1KO and A1Tg hosts were observed (p = 0.53, n = 8 per group, data not shown), MMP-9 protein levels were a significant 1.8-fold lower (p < 0.05) in tumors from A1Tg mice (Fig. 8A) and displayed decreased levels (>2-fold, p < 0.05) of enzymatic activity (Fig. 8B). ApoA1 thus appears to inhibit the level and activation of MMP-9 in the tumor microenvironment. In support of this, HDL inhibited the expression of Mmp9 in bone marrow-derived macrophages from WT mice (supplemental Table 1). View larger version: Download as PowerPoint Slide FIGURE 8. Reduced MMP-9 activity in primary tumors from A1Tg mice. Day 7 B16F10L-luciferase subcutaneous tumors were homogenized for whole cell protein extract as described under “Experimental Procedures.” A, levels of MMP-9 protein detected by Western blot with β-actin as loading control. B, MMP-9 enzyme activity in tumor extracts was assayed as described under “Experimental Procedures.” Horizontal bars in A and B represent the median of the data series. n = number of animals.

ApoA1 Suppresses Expression of Survivin in the Tumor Bed Tumors develop mechanisms to overcome death signals initiated by host cytotoxic immune cells (44). Survivin is a member of the inhibitor of apoptosis protein family of proteins with both anti-apoptotic and cell cycle promoting activities (44). In melanoma, higher cellular levels of survivin, and in particular nuclear localization of the protein, are associated with increased tumor aggressiveness and poorer patient outcome (45). Conversely, survivin down-regulation sensitizes melanoma cells to apoptosis (45). Examination of survivin protein levels and intracellular localization within day 7 primary melanoma tumors from A1Tg and A1KO mice showed survivin was expressed at a 2.7-fold lower level in A1Tg mice (Fig. 9A) with ∼24% of nuclei staining positive, as compared with ∼50% of the nuclei in the A1KO group (Fig. 9B). Tumors from A1KO mice showed higher levels of survivin in both non-nuclear and nuclear compartments (3- and 2.4-fold, respectively, p < 0.05 for each; data not shown). The higher protein levels of survivin in tumors from A1KO relative to A1Tg animals paralleled a higher survivin mRNA level observed in this group (1.4-fold, p = 0.006, data not shown). These results were also consistent with observed inhibitory effects of HDL on Birc5 (survivin) in bone marrow-derived macrophages in culture (supplemental Table 1). View larger version: Download as PowerPoint Slide FIGURE 9. Reduced survivin expression in primary tumors from A1Tg mice. A, primary day 7 B16F10L-luciferase tumors were subjected to immunohistochemistry for survivin as described under “Experimental Procedures.” Images were captured and quantified for positive survivin staining (A) and number of nuclei positive for survivin (B) as described under “Experimental Procedures.”