Markus Puhlmann

The role of interleukin 1 in growth and metastasis of human cancer xenografts.

BACKGROUND: Interleukin 1 (IL-1) is a pluripotent cytokine that promotes angiogenesis, tumor growth, and metastasis in experimental models; its presence in some human cancers is associated with aggressive tumor biology. The purpose of these studies was to characterize the role of IL-1 in human cancers and determine if inhibition of IL-1 via its receptor antagonist, IL-1Ra, alters tumor growth and metastatic potential. METHODS: IL-1 mRNA or protein levels were determined in clinical tumor samples, cancer cell lines, and xenografts using quantitative reverse transcription-PCR or ELISA. Biological activity of tumor-derived IL-1 protein was shown via induction of permeability across endothelial cell monolayers. The effects of recombinant IL-1Ra on tumor lines in culture (cell proliferation and IL-8 secretion) and in xenograft models (tumor growth, metastatic potential, and intratumoral levels of IL-8 and VEGF) were characterized. The effects of IL-1Ra-mediated regression of xenograft growth on angiogenic proteins (IL-8 and VEGF) were evaluated in an IL-1-producing melanoma (SMEL) xenograft model. RESULTS: IL-1 mRNA was highly expressed in more than half of all tested metastatic human tumor specimens including non-small-cell lung carcinoma, colorectal adenocarcinoma, and melanoma tumor samples. Constitutive IL-1 mRNA expression was identified in several cancer cell lines; tumor supernatant from these cell lines produced a significant increase in endothelial cell monolayer permeability, a hallmark event in early angiogenesis, in an IL-1-dependent manner. Moreover, systemic recombinant IL-1Ra resulted in significant inhibition of xenograft growth and neovessel density of IL-1-producing, but not non-IL-1-producing, tumor cell lines. Subsequent analysis of SMEL, a melanoma cell line with constitutive IL-1 production, showed that neither exogenous IL-1 nor IL-1Ra altered tumor cell proliferation rates in vitro. Gene expression analyses of IL-1Ra-treated SMEL xenografts showed a >3-fold down-regulation of 100 genes compared with control including a marked down-regulation of IL-8 and VEGF. CONCLUSIONS: These data show that the IL-1 gene is frequently expressed in metastases from patients with several types of human cancers. IL-1Ra inhibits xenograft growth in IL-1-producing tumors but has no direct antiproliferative effects in vitro; decreased tumor levels of IL-8 and VEGF may be an early surrogate of IL-1Ra-mediated antitumor activity. IL-1Ra may have a role alone or with other agents in the treatment of human cancers.

Vaccinia as a vector for tumor-directed gene therapy: Biodistribution of a thymidine kinase-deleted mutant

Tumor-directed gene therapy, such as “suicide gene” therapy, requires high levels of gene expression in a high percentage of tumor cells in vivo to be effective. Current vector strategies have been ineffective in achieving these goals. This report introduces the attenuated (thymidine kinase (TK)-negative) replication-competent vaccinia virus (VV) as a potential vector for tumor-directed gene therapy by studying the biodistribution of VV in animal tumor models. A TK-deleted recombinant VV (Western Reserve strain) expressing luciferase on a synthetic promoter was constructed. Luciferase activity was measured in vitro after transduction of a variety of human and murine tumor cell lines and in vivo after intraperitoneal (i.p.) delivery in C57BL/6 mice with 7-day i.p. tumors (106 MC-38 cells). Three other in vivo tumor models were examined for tumor-specific gene expression after intravenous delivery of VV (human melanoma in nude mice, adenocarcinoma liver metastasis in immunocompetent mice, and subcutaneous sarcoma in the rat). In addition, a replication-incompetent vaccinia (1 μg of psoralen and ultraviolet light, 365 nm, 4 minutes) was tested in vitro and in vivo and compared with active virus. Luciferase activity in i.p. tumors at 4 days after i.p. injection of VV was >7000-fold higher than lung, >3000-fold higher than liver, and >250-fold higher than ovary. In addition, intravenous injection of VV resulted in markedly higher tumor luciferase activity compared with any other organ in every model tested (up to 188,000-fold higher than liver and 77,000-fold higher than lung). Inactivation of the virus resulted in negligible gene expression in vivo. In summary, VV has a high transduction efficiency in tumor cells with high levels of gene expression. The results suggest a selective in vivo replication of TK-deleted VV in tumor cells. Replication competent, TK-deleted VV appears to be an ideal vector for testing the in vivo delivery of toxic genes to tumor cells.

Gene-expression profiling of the response of peripheral blood mononuclear cells and melanoma metastases to systemic IL-2 administration

Backgroundlnterleukin-2 (IL-2) has direct pluripotent effects on cells with immune and inflammatory function. Which of these effects has a critical role in mediating tumor regression remains enigmatic. In this study, we compared early changes in transcriptional profiles of circulating mononuclear cells with those occurring within the microenvironment of melanoma metastases following systemic IL-2 administration.ResultsThe results suggest that the immediate effects of IL-2 administration on the tumor microenvironment is transcriptional activation of genes predominantly associated with monocyte cell function; minimal effects were noted on migration, activation and proliferation of T cells. However, production of chemokines and markers of adhesion and migration within few hours of IL-2 administration may be responsible for a secondary recruitment of immune cells to the tumor site later.ConclusionOur results suggest that IL-2 induces inflammation at tumor sites with three predominant secondary effects: activation of antigen-presenting monocytes; massive production of chemoattractants that may recruit other immune cells to the tumor (including MIG and PARC, which are specific for T cells); and activation of cytolytic mechanisms in monocytes (calgranulin, grancalcin) and NK cells (NKG5, NK4).

Thymidine kinase-deleted vaccinia virus expressing purine nucleoside phosphorylase as a vector for tumor-directed gene therapy.

Tumor-directed gene therapy faces many obstacles. Lack of tissue targeting and low in vivo transduction efficiency represent some of the limitations for a successful therapeutic outcome. A thymidine kinase-deleted mutant vaccinia virus has been shown in marker studies to replicate selectively in tumor tissue in animal models. Purine nucleoside phosphorylase (PNP), from E. coli, converts the nontoxic prodrug 6-methylpurine deoxyriboside (6-MPDR) to the toxic purine 6-methylpurine. In this study, we investigated the cytotoxic properties of PNP, expressed by an optimized synthetic early/late promoter in a vaccinia virus (vMPPNP). In vitro cytotoxicity of psoralen-inactivated vMPPNP (1 microg of psoralen, 4 min of LWUV [365 nm]) at the maximum tolerated dose (MTD) of 6-MPDR (80 microM) reduced cell viability by day 3 to 1.7%. At an MOI of 0.002, replication-competent vMPPNP and 6-MPDR (80 microM) caused reduction of cell viability to 19.8% within 4 days. Furthermore, there was complete abrogation of viral replication after intracellular conversion of prodrug into the active toxin. The potency of such a system was similar among all histologies tested. Finally, the cytotoxic efficacy has been shown to be more rapid and complete than that of cytosine deaminase (CD), a more established enzyme/prodrug system. When virus was delivered intraperitoneally into athymic mice with hepatic metastases, followed by administration of prodrug, there was a significant prolongation of survival and a 30% cure rate. In summary, owing to its tumor-targeting capabilities, high transduction efficiency, and high gene expression, a vaccinia virus expressing PNP could prove to be a potent and valuable vector for tumor-targeted gene therapy.

Direct evidence for rapid and selective induction of tumor neovascular permeability by tumor necrosis factor and a novel derivative, colloidal gold bound tumor necrosis factor

Tumor necrosis factor (TNF) causes regression of advanced cancers when used in isolation perfusion with melphalan; evidence suggests these effects are mediated via selective yet uncharacterized actions on tumor neovasculature. A novel derivative, colloidal gold bound TNF (cAu‐TNF) has been shown to have similar antitumor effects as native TNF with less systemic toxicity in mice. These studies were done to determine their effects on tumor neovasculature, using in vivo video microscopy. Female C57BL/6 mice bearing 20 mm2 MC38 or LLC tumors that are TNF sensitive and resistant tumors, respectively, had dorsal skinfold chambers implanted. The rate of interstitial accumulation of Texas red fluorescently labeled albumin in tumor and normal vasculature was measured after intravenous TNF, cAu‐TNF or PBS. Changes in interstitial fluorescent intensity over time were quantified as a reflection of alterations in vascular permeability. MC38 bearing mice treated with TNF or cAu‐TNF demonstrated a rapid, selective and significant increase in tracer accumulation in areas of neovasculature compared to those of normal vasculature. Experiments in LLC tumor bearing mice showed similar results. Monoclonal antibody against tissue factor partially abrogated the effects of TNF on MC38 neovasculature. These data provide direct evidence that TNF and cAu‐TNF selectively and rapidly alter permeability in tumor neovasculature; a phenomenon that may be exploited to enhance selective delivery of chemotherapeutics to tumor.

Regional versus Systemic Delivery of Recombinant Vaccinia Virus as Suicide Gene Therapy for Murine Liver Metastases

OBJECTIVE Specific and efficient tumor-targeted gene delivery is the major goal for successful cancer gene therapy. SUMMARY BACKGROUND DATA A recombinant thymidine kinase-deleted vaccinia virus (vv) encoding the firefly luciferase (luc) reporter gene or the prodrug converter gene cytosine deaminase (CD) was constructed. The authors compared the extent, duration, and pattern of transgene (luc) expression in vivo after portal venous, intraperitoneal, or intravenous virus administration and survival after treatment with the vv containing CD followed by the prodrug 5-fluorocytosine (5-FC) in a murine model of disseminated liver metastases from colon cancer. METHODS Recombinant vv containing the luc transgene within the thymidine kinase locus was administered to mice with isolated liver metastases from an MC38 adenocarcinoma. Transgene expression was determined in tumor and organs at various time points. Tumor-bearing mice were treated with recombinant vv containing CD and 5-FC or with appropriate controls and followed for survival. RESULTS Tumor-specific gene delivery was achieved irrespective of administration route, with gene expression in tumors increased by up to 100,000-fold compared with normal tissues. There was significantly increased transgene expression in tumor after portal venous or intraperitoneal virus administration (p = 0.001 vs. systemic). Treatment using a CD-expressing vv and systemic 5-FC resulted in a significant survival benefit in all treatment groups compared with controls (p < 0.007); there was no additional benefit for portal venous or intraperitoneal virus administration. CONCLUSIONS Suicide gene therapy using vv with the CD/5-FC system leads to tumor-specific gene expression and improved survival and can result in cure of established liver metastases.

Interleukin-1β induced vascular permeability is dependent on induction of endothelial Tissue Factor (TF) activity

IL-1β is a pleotropic cytokine that may mediate increased procoagulant activity and permeability in endothelial tissue during inflammatory conditions. The procoagulant effects of IL-1β are mediated through induction of tissue factor (TF) but its alterations on vascular permeability are not well characterized. We found that IL-1β induced a rapid and dose-dependent increase in TF activity in human umbilical vein endothelial cells (ECs) under routine culture conditions. However, IL-1β caused a rapid and marked increase in permeability across confluent EC monolayers using a two-compartment in vitro model only in the presence of factor VIII-deficient plasma that was completely abrogated by neutralizing anti-TF antibody pre-treatment. In vitro permeability was associated with loss of EC surface expression of VE-cadherin and contraction of F-actin cytoskeletal elements that resulted in EC intercellular gap formation. These data demonstrate that IL-1β induces marked changes in permeability across activated endothelium via a TF dependent mechanism and suggest that modulation of TF activity may represent a strategy to treat various acute and chronic inflammatory conditions mediated by this cytokine.

Alterations in tumor necrosis factor-induced endothelial cell procoagulant activity by hyperthermia

TNF is a cytokine with potent antitumor activity in murine models and when administered clinically via regional perfusion. There is substantial evidence that this antitumor activity depends in large part on TNFs procoagulant effect on tumor neovasculature, which is mediated by induction of endothelial cell tissue factor (TF), a component of the extrinsic clotting cascade. In regional perfusion of a cancer‐bearing limb or organ, TNF is always administered under hyperthermic temperatures; however, little is known about the effect of hyperthermia on TNF‐mediated procoagulant activity in endothelium. We examined the effects of hyperthermia on TNF‐mediated procoagulant activity in human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to TNF at normothermic (37°C) and hyperthermic (41°C) temperatures for 90 min, then assayed for clotting activity, TF protein production and mRNA production of TF and tissue factor pathway inhibitor‐2 (TFPI‐2), an endogenous inducible inhibitor of TF activity in HUVECs. TNF treatment at 41°C significantly reduced clotting activity, TF protein and mRNA as well as TFPI‐2 mRNA compared to treatment at 37°C. These data show that hyperthermia significantly reduces the procoagulant effects of TNF on endothelial tissue compared to normothermia, which may have important clinical implications for the use of TNF in regional perfusion.