Altered expression of 15-hydroxyprostaglandin dehydrogenase in tumor-infiltrated CD11b myeloid cells: a mechanism for immune evasion in cancer.

Many cancers are known to produce high amounts of PGE(2), which is involved in both tumor progression and tumor-induced immune dysfunction. The key enzyme responsible for the biological inactivation of PGE(2) in tissue is NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). It is well established that cancer cells frequently show down-regulated expression of 15-PGDH, which plays a major role in catabolism of the PGE(2). Here we demonstrate that tumor-infiltrated CD11b cells are also deficient for the 15-PGDH gene. Targeted adenovirus-mediated delivery of 15-PGDH gene resulted in substantial inhibition of tumor growth in mice with implanted CT-26 colon carcinomas. PGDH-mediated antitumor effect was associated with attenuated tumor-induced immune suppression and substantially reduced secretion of immunosuppressive mediators and cytokines such as PGE(2), IL-10, IL-13, and IL-6 by intratumoral CD11b cells. We show also that introduction of 15-PGDH gene in tumor tissue is sufficient to redirect the differentiation of intratumoral CD11b cells from immunosuppressive M2-oriented F4/80(+) tumor-associated macrophages (TAM) into M1-oriented CD11c(+) MHC class II-positive myeloid APCs. Notably, the administration of the 15-PGDH gene alone demonstrated a significant therapeutic effect promoting tumor eradication and long-term survival in 70% of mice with preestablished tumors. Surviving mice acquired antitumor T cell-mediated immune response. This study for the first time demonstrates an important role of the 15-PGDH in regulation of local antitumor immune response and highlights the potential to be implemented to enhance the efficacy of cancer therapy and immunotherapy

Transcriptional targeting of primary and metastatic tumor neovasculature by an adenoviral type 5 roundabout4 vector in mice.

New approaches targeting metastatic neovasculature are needed. Payload capacity, cellular transduction efficiency, and first-pass cellular uptake following systemic vector administration, motivates persistent interest in tumor vascular endothelial cell (EC) adenoviral (Ad) vector targeting. While EC transductional and transcriptional targeting has been accomplished, vector administration approaches of limited clinical utility, lack of tumor-wide EC expression quantification, and failure to address avid liver sequestration, challenged prior work. Here, we intravenously injected an Ad vector containing 3 kb of the human roundabout4 (ROBO4) enhancer/promoter transcriptionally regulating an enhanced green fluorescent protein (EGFP) reporter into immunodeficient mice bearing 786-O renal cell carcinoma subcutaneous (SC) xenografts and kidney orthotopic (KO) tumors. Initial experiments performed in human coxsackie virus and adenovirus receptor (hCAR) transgenic:Rag2 knockout mice revealed multiple ECs with high-level Ad5ROBO4-EGFP expression throughout KO and SC tumors. In contrast, Ad5CMV-EGFP was sporadically expressed in a few tumor vascular ECs and stromal cells. As the hCAR transgene also facilitated Ad5ROBO4 and control Ad5CMV vector EC expression in multiple host organs, follow-on experiments engaged warfarin-mediated liver vector detargeting in hCAR non-transgenic mice. Ad5ROBO4-mediated EC expression was undetectable in most host organs, while the frequencies of vector expressing intratumoral vessels and whole tumor EGFP protein levels remained elevated. In contrast, AdCMV vector expression was only detectable in one or two stromal cells throughout the whole tumor. The Ad5ROBO4 vector, in conjunction with liver detargeting, provides tractable genetic access for in-vivo EC genetic engineering in malignancies

Warfarin liver detargeting enhances tumor neovascular endothelial cell reporter expression of the Ad5ROBO4 vector.

<p>A. Ad5ROBO4 produced easily detectable scattered tumor endothelial cell EGFP immunofluorescence in both kidney orthotopic and subcutaneous 786-O tumors in vehicle-treated <i>Rag2^−/−</i> mice. B. Warfarin pretreatment markedly enhanced the multiplicity of tumor endothelial cell reporter gene expression within both orthotopic and subcutaneous tumors in Ad5ROBO-injected mice. C. Ad5CMV injection failed to produce detectable tumor EC expression in vehicle-treated, or D. warfarin-treated mice. A–D: tumors from the same mice as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083933#pone-0083933-g005" target="_blank">Figure 5</a>. Magnifications: 40X first and fourth columns, 100X second and fifth columns, and 400X third and sixth columns. Red: endomucin/CD31, Green: EGFP immunofluorescence, Blue: DAPI.</p

Warfarin pretreatment enhances tumor Ad5ROBO4 vector expression.

<p>A. Multiorgan immunoblot of vehicle (left lanes) or warfarin (right lanes) pretreated <i>Rag2^−/−</i> mice injected with 1.0×10¹¹ vp of Ad5ROBO4. B. Densitometry of A revealed that vehicle pretreatment was associated with robust liver, detectable splenic, and trace to undetectable expression in all other sampled organs. Warfarin pretreatment produced a 2.5-fold increased splenic and a 3-fold decreased liver expression while all other organs still evidenced trace to undetectable expression. C. Immunoblot and densitometry of liver and tumor EGFP, VE-cadherin, and β-tubulin expression in vehicle (left lanes) or warfarin (right lanes) from the same pretreated, Ad5ROBO4-injected mice as in A and B. EGFP densitometry normalized to VE-cadherin, revealed a 4.7-fold decrease in liver and 2-fold increase in increase KO and SC tumor expression produced by warfarin pretreatment. A–C: representative immunoblots from n = 2 mice from 2 independent experiments.</p

Endogenous ROBO4 upregulation despite lower vascular density in orthotopic and xenograft tumors.

<p>A. Immunofluorescence of the vascular endothelium in liver (upper panel) and 786-O human renal cell carcinoma (RCC) subcutaneous xenograft tumor (lower panel). B. Vascular area analysis of liver (Li), kidney orthotopic (KO) tumors, and subcutaneous (SC) xenograft tumors (n = 6 mice analyzed). C. Immunoblot of endogenous ROBO4 and the endothelial cell specific VE-cadherin from liver, kidney orthotopic and subcutaneous xenograft 786-O RCC tumors, and from the derivative 786-O cells grown in culture. D. Densitometry analysis of VE-cadherin/tubulin ratio from C mirrors the vascular area determination in B. E. Densitometry analysis of endogenous ROBO4 normalized to VE-cadherin expression reveals a 1.4- to 2-fold increase in SC and KO tumors compared to liver. C–E: Immunoblot and densitometry was repeated twice with two independent sets of protein extracts from two different tumor-bearing mice with essentially the same results. A. Magnification: 100X, Red: endomucin/CD31 antibody cocktail, Blue: DAPI. B. *p<0.05, one way ANOVA with Tukey's correction, mean ± SD.</p

Adenoviral Type 5 (Ad5) vector expression in a host organ panel in tumor bearing immunodeficient <i>Rag2^−/−</i>, and <i>hCAR:Rag2^−/−</i> composite mice.

<p>Host organ EGFP reporter expression following intravenous injection of 1.0×10¹¹ viral particles (vp) of either (A, B) Ad5ROBO4-EGFP (ROBO4) or (C, D) Ad5CMV-EGFP (CMV) vectors. A. Ad5ROBO4 vector expression in <i>Rag2^−/−</i> mice is widespread but focal in liver endothelial cells (ECs), and rarely detectable in single splenic microvessels. All other organs are negative for vector reporter expression. B. Multiorgan, EC restricted vascular expression is evident in Ad5ROBO4-injected <i>hCAR:Rag2^−/−</i> composite mice. C. Ad5CMV vector expression in <i>Rag2^−/−</i> mice is detectable in liver hepatocytes, sporadic splenic (inflammatory) and adrenal cells. D. Ad5CMV vector expression in <i>hCAR:Rag2^−/−</i> mice is complex, hepatocyte localized, but decreased in frequency compared to C in liver, mixed inflammatory and endothelial cell localized in spleen, and EC localized in all other organs including brain and skin. A and B: n = 5–6 mice, combined from 3–6 independent experiments, C and D: n = 3–4 mice combined from 3–4 independent experiments Magnification: 100X, Red: endomucin/CD31, Green: EGFP immunofluorescence, Blue: DAPI.</p

Warfarin pretreatment “detargets” the liver without producing multiorgan Ad5ROBO4 vector expression.

<p>A. Ad5ROBO4 injection into vehicle-treated <i>Rag2^−/−</i> mice produced vascular EC expression predominantly in liver and spleen. B. Warfarin pretreatment, 5 mg/kg, on day −3 and −1 prior to Ad5ROBO4 injection vector injection increased the frequency of splenic and lung EC expression and produced sporadic, infrequent expression in kidney and heart. Liver expression was present but diminished. C. AdCMV injection into vehicle treated mice predominantly produced hepatocyte expression with focal RES cell splenic and scattered lung expression. D. Warfarin pretreatment prior to AdCMV injection markedly decreased the frequency of hepatocyte EGFP expression, while increasing sporadic splenic, and lung expression, inducing focal adrenal cellular and rare muscle and skin EC expression. A, B and S5: Representative images from n = 5 mice from 2 independent experiments, C and D: n = 3 mice from 3 independent experiments. 1×10¹¹ vp were injected in each group. Magnification 100X; Red: endomucin/CD31, Green: EGFP immunofluorescence, Blue: DAPI.</p

Semiquantitative immunoblotting reveals differential Ad5ROBO4 reporter expression in tumor compared to liver.

<p>A. Immunoblot of EGFP, VE-cadherin, and β-tubulin loading controls in tissue protein extracts from <i>hCAR:Rag2^−/−</i> mice injected with either Ad5ROBO4, left three lanes, or Ad5CMV, right three lanes. B. and C. Densitometry analysis of Ad5ROBO4 vector EGFP expression normalized to either VE-cadherin or β-tubulin. D. Densitometry of AdCMV vector EGFP expression. As AdCMV expression was hepatocyte specific, this blot was only normalized to β-tubulin. A–D: Representative immunoblots from n = 4 mice injected with either Ad5ROBO 4 or Ad5CMV vectors. Li: liver, KO: kidney orthotopic tumor, SC: subcutaneous tumor.</p

Vascular restricted ROBO4-directed reporter expression in kidney orthotopic and subcutaneous xenograft tumors.

<p>The tumors were harvested from the same Ad vector injected mice whose host organs were depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083933#pone-0083933-g002" target="_blank">Figure 2</a>. A. Ad5ROBO4 vector expression in <i>Rag2^−/−</i> mice is sporadic yet easily detectable in vascular ECs within tumors in either orthotopic or subcutaneous microenvironments. B. The <i>hCAR</i> transgene markedly increases Ad5ROBO4 vector expression throughout both the orthotopic and subcutaneous tumors following injection into <i>hCAR:Rag2^−/−</i> mice. C. Ad5CMV expression in <i>Rag2^−/−</i> mice is undetectable in either orthotopic or subcutaneous tumors. D. Isolated intratumoral endothelial cell reporter expression in <i>hCAR:Rag2^−/−</i> mice. White line: kidney-tumor boundary; Arrows: glomerular tufts. Magnifications: 40X, first and fourth columns, and 200X, second and fifth columns, 400X, third and sixth columns. Red: endomucin/CD31, Green: EGFP immunofluorescence, Blue: DAPI.</p