The cardioprotective protein apolipoprotein A1 promotes potent anti-tumorigenic effects

Abstract

Here, we show that apolipoprotein A1 (apoA1), the major protein component of high density lipoprotein (HDL), through both innate and adaptive immune processes, potently suppresses tumor growth and metastasis in multiple animal tumor models, including the aggressive B16F10L murine malignant melanoma model. Mice expressing the human apoA1 transgene (A1Tg) exhibited increased infiltration of CD11b(+) F4/80(+) macrophages with M1, anti-tumor phenotype, reduced tumor burden and metastasis, and enhanced survival. In contrast, apoA1-deficient (A1KO) mice showed markedly heightened tumor growth and reduced survival. Injection of human apoA1 into A1KO mice inoculated with tumor cells remarkably reduced both tumor growth and metastasis, enhanced survival, and promoted regression of both tumor and metastasis burden when administered following palpable tumor formation and metastasis development. Studies with apolipoprotein A2 revealed the anti-cancer therapeutic effect was specific to apoA1. In vitro studies ruled out substantial direct suppressive effects by apoA1 or HDL on tumor cells. Animal models defective in different aspects of immunity revealed both innate and adaptive arms of immunity contribute to complete apoA1 anti-tumor activity. This study reveals a potent immunomodulatory role for apoA1 in the tumor microenvironment, altering tumor-associated macrophages from a pro-tumor M2 to an anti-tumor M1 phenotype. Use of apoA1 to redirect in vivo elicited tumor-infiltrating macrophages toward tumor rejection may hold benefit as a potential cancer therapeutic.

Keywords: Adaptive Immunity; Apolipoprotein A1 Therapy; Cancer; Cancer Biology; Cancer Therapy; High Density Lipoprotein (HDL); Innate Immunity; Matrix Metalloproteinase 9 (MMP-9); Melanoma; Tumor-suppressive Infiltrating Leukocytes.

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.

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.

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.

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.”

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.

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).

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 G₁ (red), with each subsequent smaller generation represented as G₂–G₆ (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.

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.

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.”

FIGURE 10.

ApoA1 promotes the infiltration of F4/80⁺ macrophages into tumor bed. Primary day 7 B16F10L-luciferase tumors were subjected to immunohistochemistry for CD11b⁺ or F4/80⁺ (red) and nuclei stained with DAPI (blue). The entire tumor area was captured with a ×20 lens, and digitized images were assessed for positive staining as described under “Experimental Procedures.” Representative images for anti CD11b⁺ or F4/80⁺ staining of tumors are shown in A. Top panels were stained for CD11b⁺, and lower panels were stained for F4/80⁺. Scale bars represent 500 μm. Insets from A are shown at a higher magnification in B where scale bars represent 75 μm. CD11b protein expression was quantified as described under “Experimental Procedures.” C, data indicate the numbers and genotypes of mice.

FIGURE 11.

F4/80⁺ cells recovered from tumor-bearing A1Tg mice are cytotoxic to tumor cells in vitro. F4/80⁺ effector (E) cells immunopurified from elicited peritoneal exudates of day 19 tumor-bearing animals were cocultured with B16F10L target (T) cells in vitro, and cell viability was assessed after 3 days as described under “Experimental Procedures.”

FIGURE 12.

ApoA1 promotes tumor infiltration of cytotoxic CD8 T cells. FACS analysis of pooled tumors (per genotype; six sites/animal; n = number of animals). Day 15 tumors were digested and labeled with antibodies to surface antigens as described under “Experimental Procedures.” TILs were identified as CD45.2⁺, and the percent of TILs that were either CD3⁺CD8⁺ or CD3⁺CD4⁺ was quantified by flow cytometry as described under “Experimental Procedures.”

FIGURE 13.

Net functional effects of apoA1/HDL in tumor microenvironment. Overall scheme describing role of apoA1/HDL in the tumor microenvironment in melanoma tumor biology.