Sven A. Lang

Right Portal Vein Ligation Combined With In Situ Splitting Induces Rapid Left Lateral Liver Lobe Hypertrophy Enabling 2-Staged Extended Right Hepatic Resection in Small-for-Size Settings

Objective:To evaluate a new 2-step technique for obtaining adequate but short-term parenchymal hypertrophy in oncologic patients requiring extended right hepatic resection with limited functional reserve. Background:Patients presenting with primary or metastatic liver tumors often face the dilemma that the remaining liver tissue may not be sufficient. Preoperative portal vein embolization has thus far been established as the standard procedure for achieving resectability. Methods:Two-staged hepatectomy was performed in patients who preoperatively appeared to be marginally resectable but had a tumor-free left lateral lobe. Marginal respectability was defined as a left lateral lobe to body weight ratio of less than 0.5. In the first step, surgical exploration, right portal vein ligation (PVL), and in situ splitting (ISS) of the liver parenchyma along the falciform ligament were performed. Computed tomographic volumetry was performed before ISS and before completion surgery. Results:The study included 25 patients with primary liver tumors (hepatocellular carcinoma: n = 3, intrahepatic cholangiocarcinoma: n = 2, extrahepatic cholangiocarcinoma: n = 2, malignant epithelioid hemangioendothelioma: n = 1, gallbladder cancer: n = 1 or metastatic disease [colorectal liver metastasis]: n = 14, ovarian cancer: n = 1, gastric cancer: n = 1). Preoperative CT volumetry of the left lateral lobe showed 310 mL in median (range = 197–444 mL). After a median waiting period of 9 days (range = 5–28 days), the volume of the left lateral lobe had increased to 536 mL (range = 273–881 mL), representing a median volume increase of 74% (range = 21%–192%) (P < 0.001). The median left lateral liver lobe to body weight ratio was increased from 0.38% (range = 0.25%–0.49%) to 0.61% (range = 0.35–0.95). Ten of 25 patients (40%) required biliary reconstruction with hepaticojejunostomy. Rapid perioperative recovery was reflected by normalization of International normalized ratio (INR) (80% of patients), creatinine (84% of patients), nearly normal bilirubin (56% of patients), and albumin (64% of patients) values by day 14 after completion surgery. Perioperative morbidity was classified according to the Dindo-Clavien classification of surgical complications: grade I (12 events), grade II (13 events), grade III (14 events, III a: 6 events, III b: 8 events), grade IV (8 events, IV a: 3 events, IV b: 5 events), and grade V (3 events). Sixteen patients (68%) experienced perioperative complications. Follow-up was 180 days in median (range: 60–776 days) with an estimated overall survival of 86% at 6 months after resection. Conclusions:Two-step hepatic resection performing surgical exploration, PVL, and ISS results in a marked and rapid hypertrophy of functional liver tissue and enables curative resection of marginally resectable liver tumors or metastases in patients that might otherwise be regarded as palliative.

Mesenchymal stem cells can induce long-term acceptance of solid organ allografts in synergy with low-dose mycophenolate.

The induction of tolerance towards allogeneic solid organ grafts is one of the major goals in transplantation medicine. Mesenchymal stem cells (MSC) inhibit the immune response in vitro, and thus are promising candidate cells to promote acceptance of transplanted organs in vivo. Such novel approaches of tolerance induction are needed since, to date, graft acceptance can only be maintained through life-long treatment with unspecific immunosuppressants that are associated with toxic injury, opportunistic infections and malignancies. We demonstrate that donor-derived MSC induce long-term allograft acceptance in a rat heart transplantation model, when concurrently applied with a short course of low-dose mycophenolate. This tolerogenic effect of MSC is at least partially mediated by the expression of indoleamine 2,3-dioxygenase (IDO), demonstrated by the fact that blocking of IDO with 1-methyl tryptophan (1-MT) abrogates graft acceptance. Moreover we hypothesize that MSC interact with dendritic cells (DC) in vivo, because allogeneic MSC are rejected in the long-term but DC acquire a tolerogenic phenotype after applying MSC. In summary, we demonstrate that MSC constitute a promising tool for induction of non-responsiveness in solid organ transplantation that warrants further investigation in clinical trials.

Mammalian target of rapamycin is activated in human gastric cancer and serves as a target for therapy in an experimental model

The mammalian target of rapamycin (mTOR) has become an interesting target for cancer therapy through its influence on oncogenic signals, which involve phosphatidylinositol‐3‐kinase and hypoxia‐inducible factor‐1α (HIF‐1α). Since mTOR is an upstream regulator of HIF‐1α, a key mediator of gastric cancer growth and angiogenesis, we investigated mTOR activation in human gastric adenocarcinoma specimens and determined whether rapamycin could inhibit gastric cancer growth in mice. Expression of phospho‐mTOR was assessed by immunohistochemical analyses of human tissues. For in vitro studies, human gastric cancer cell lines were used to determine S6K1, 4E‐BP‐1 and HIF‐1α activation and cancer cell motility upon rapamycin treatment. Effects of rapamycin on tumor growth and angiogenesis in vivo were assessed in both a subcutaneous tumor model and in an experimental model with orthotopically grown tumors. Mice received either rapamycin (0.5 mg/kg/day or 1.5 mg/kg/day) or diluent per intra‐peritoneal injections. In addition, antiangiogenic effects were monitored in vivo using a dorsal‐skin‐fold chamber model. Immunohistochemical analyses showed strong expression of phospho‐mTOR in 60% of intestinal‐ and 64% of diffuse‐type human gastric adenocarcinomas. In vitro, rapamycin‐treatment effectively blocked S6K1, 4E‐BP‐1 and HIF‐1α activation, and significantly impaired tumor cell migration. In vivo, rapamycin‐treatment led to significant inhibition of subcutaneous tumor growth, decreased CD31‐positive vessel area and reduced tumor cell proliferation. Similar significant results were obtained in an orthotopic model of gastric cancer. In the dorsal‐skin‐fold chamber model, rapamycin‐treatment significantly inhibited tumor vascularization in vivo. In conclusion, mTOR is frequently activated in human gastric cancer and represents a promising new molecular target for therapy.

Targeting Heat Shock Protein 90 in Pancreatic Cancer Impairs Insulin-like Growth Factor-I Receptor Signaling, Disrupts an Interleukin-6/Signal-Transducer and Activator of Transcription 3/Hypoxia-Inducible Factor-1α Autocrine Loop, and Reduces Orthotopic Tumor Growth

Purpose: Inhibitors of heat-shock protein 90 (Hsp90) may interfere with oncogenic signaling pathways, including Erk, Akt, and hypoxia-inducible factor-1α (HIF-1α). Because insulin-like growth factor-I receptor (IGF-IR) and signal transducer and activator of transcription 3 (STAT3) signaling pathways are implicated in the progression of pancreatic cancer, we hypothesized that blocking Hsp90 with geldanamycin derivates [17-allylamino-geldanamycin (17-AAG), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG)] would impair IGF-I– and interleukin-6–mediated signaling and thus reduce pancreatic tumor growth and angiogenesis in vivo. Experimental Design: Human pancreatic cancer cells (HPAF-II, L3.6pl) were used for experiments. Changes in signaling pathway activation upon Hsp90 blockade were investigated by Western blotting. Effects of Hsp90 inhibition (17-AAG) on vascular endothelial growth factor were determined by ELISA and real-time PCR. Effects of 17-DMAG (25 mg/kg; thrice a week; i.p.) on tumor growth and vascularization were investigated in a s.c. xenograft model and in an orthotopic model of pancreatic cancer. Results: 17-AAG inhibited IGF-IR signaling by down-regulating IGF-IRβ and directly impairing IGF-IR phosphorylation. Hypoxia- and IL-6–mediated activation of HIF-1α or STAT3/STAT5 were substantially inhibited by 17-AAG. Moreover, a novel IL-6/STAT3/HIF-1α autocrine loop was effectively disrupted by Hsp90 blockade. In vivo, 17-DMAG significantly reduced s.c. tumor growth and diminished STAT3 phosphorylation and IGF-IRβ expression in tumor tissues. In an orthotopic model, pancreatic tumor growth and vascularization were both significantly reduced upon Hsp90 inhibition, as reflected by final tumor weights and CD31 staining, respectively. Conclusions: Blocking Hsp90 disrupts IGF-I and IL-6–induced proangiogenic signaling cascades by targeting IGF-IR and STAT3 in pancreatic cancer, leading to significant growth-inhibitory effects. Therefore, we suggest that Hsp90 inhibitors could prove to be valuable in the treatment of pancreatic cancer.

Inhibition of heat shock protein 90 impairs epidermal growth factor-mediated signaling in gastric cancer cells and reduces tumor growth and vascularization in vivo

Oncogenic signaling through activation of epidermal growth factor receptor (EGFR), HER-2, and hypoxia inducible-factor-1α (HIF-1α) has been implicated in gastric cancer growth and angiogenesis through up-regulation of vascular endothelial growth factor (VEGF). Recently, heat shock protein 90 (Hsp90) has been identified as a critical regulator of oncogenic protein stability, including EGFR, HER-2, and HIF-1α. We hypothesized that inhibition of Hsp90 impairs EGF- and hypoxia-mediated angiogenic signaling in gastric cancer cells and consequently inhibits angiogenesis and tumor growth. In vitro, the geldanamycin derivate 17-allylamino-17-demethoxygeldanamycin (17-AAG) led to marked reduction in constitutive and inducible activation of extracellular signal-regulated kinase 1/2, Akt, and signal transducer and activator of transcription 3 and decreased nuclear HIF-1α protein. In addition, EGFR and HER-2 were down-regulated after Hsp90 inhibition. With respect to regulation of angiogenic molecules, 17-AAG significantly reduced EGF-mediated VEGF secretion. Phosphorylation of focal adhesion kinase and paxillin were both abrogated by 17-AAG, which resulted in significant impairment of cancer cell motility. Interestingly, cytotoxic effects of 17-AAG in vitro were higher on cancer cells and gastric fibroblasts than on pericytes. In vivo, the water-soluble compound 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; 25 mg/kg, thrice per week) significantly reduced s.c. xenografted tumor growth. By immunohistochemistry, 17-DMAG significantly reduced vessel area and numbers of proliferating tumor cells in sections. Furthermore, similar significant growth-inhibitory effects of 17-DMAG were achieved when administered as low-dose therapy (5 mg/kg, thrice per week). In conclusion, blocking Hsp90 disrupts multiple proangiogenic signaling pathways in gastric cancer cells and inhibits xenografted tumor growth in vivo. Hence, gastric cancer harbors attractive molecular targets for therapy with Hsp90 inhibitors, which could lead to improved efficacy of antineoplastic therapy regimens. [Mol Cancer Ther 2007;6(3):1123–32]

Targeting FGFR/PDGFR/VEGFR impairs tumor growth, angiogenesis and metastasis by effects on tumor cells, endothelial cells and pericytes in pancreatic cancer

Activation of receptor tyrosine kinases, such as fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), and VEGF receptor (VEGFR), has been implicated in tumor progression and metastasis in human pancreatic cancer. In this study, we investigated the effects of TKI258, a tyrosine kinase inhibitor to FGFR, PDGFR, and VEGFR on pancreatic cancer cell lines (HPAF-II, BxPC-3, MiaPaCa2, and L3.6pl), endothelial cells, and vascular smooth muscle cells (VSMC). Results showed that treatment with TKI258 impaired activation of signaling intermediates in pancreatic cancer cells, endothelial cells, and VSMCs, even upon stimulation with FGF-1, FGF-2, VEGF-A, and PDGF-B. Furthermore, blockade of FGFR/PDGFR/VEGFR reduced survivin expression and improved activity of gemcitabine in MiaPaCa2 pancreatic cancer cells. In addition, motility of cancer cells, endothelial cells, and VSMCs was reduced upon treatment with TKI258. In vivo, therapy with TKI258 led to dose-dependent inhibition of subcutaneous (HPAF-II) and orthotopic (L3.6pl) tumor growth. Immunohistochemical analysis revealed effects on tumor cell proliferation [bromodeoxyuridine (BrdUrd)] and tumor vascularization (CD31). Moreover, lymph node metastases were significantly reduced in the orthotopic tumor model when treatment was initiated early with TKI258 (30 mg/kg/d). In established tumors, TKI258 (30 mg/kg/d) led to significant growth delay and improved survival in subcutaneous and orthotopic models, respectively. These data provide evidence that targeting FGFR/PDFGR/VEGFR with TKI258 may be effective in human pancreatic cancer and warrants further clinical evaluation. Mol Cancer Ther; 10(11); 2157–67. ©2011 AACR.

Tumor development in murine ulcerative colitis depends on MyD88 signaling of colonic F4/80 + CD11b high Gr1 low macrophages

Patients with prolonged ulcerative colitis (UC) frequently develop colorectal adenocarcinoma for reasons that are not fully clear. To analyze inflammation-associated colonic tumorigenesis, we developed a chronic form of oxazolone-induced colitis in mice that, similar to UC, was distinguished by the presence of IL-13-producing NKT cells. In this model, the induction of tumors using azoxymethane was accompanied by the coappearance of F4/80+CD11b(high)Gr1(low) M2 macrophages, cells that undergo polarization by IL-13 and are absent in tumors that lack high level IL-13 production. Importantly, this subset of macrophages was a source of tumor-promoting factors, including IL-6. Similar to dextran sodium sulfate-induced colitis, F4/80+CD11b(high)Gr1(intermediate) macrophages were present in the mouse model of chronic oxazolone-induced colitis and may influence tumor development through production of TGF-β1, a cytokine that inhibits tumor immunosurveillance. Finally, while robust chronic oxazolone-induced colitis developed in myeloid differentiation primary response gene 88-deficient (Myd88-/-) mice, these mice did not support tumor development. The inhibition of tumor development in Myd88-/- mice correlated with cessation of IL-6 and TGF-β1 production by M2 and F4/80+CD11b(high)Gr1(intermediate) macrophages, respectively, and was reversed by exogenous IL-6. These data show that an UC-like inflammation may facilitate tumor development by providing a milieu favoring development of MyD88-dependent tumor-supporting macrophages.

Inhibition of insulin-like growth factor-I receptor (IGF-IR) using NVP-AEW541, a small molecule kinase inhibitor, reduces orthotopic pancreatic cancer growth and angiogenesis.

The insulin-like growth factor-I receptor (IGF-IR) is frequently overexpressed and constitutively activated in pancreatic cancer, thus representing a promising target for therapy. We investigated the impact of a novel inhibitor of IGF-IR (NVP-AEW541) on signalling and growth of pancreatic cancer. Human pancreatic cancer cells and endothelial cells were employed, and effects of NVP-AEW541 on signalling pathways investigated by Western blotting. NVP-AEW541 diminished the activation of IGF-IR, IRS-1, Erk, Akt and STAT3. Furthermore, NVP-AEW541 reduced cancer cell proliferation and abrogated migratory effects of IGF-I. NVP-AEW541 elicited a direct effect on endothelial cells in terms of reducing endothelial cell migration. In vivo, treatment of mice with NVP-AEW541 significantly reduced orthotopic pancreatic tumour growth, vascularisation, and VEGF expression. Interestingly, NVP-AEW541 lowered serum levels of IGF-binding-protein-3 (IGFBP-3). In conclusion, the IGF-IR inhibitor NVP-AEW541 effectively disrupts IGF-I signalling and reduces pancreatic tumour growth. Hence, blocking IGF-IR could prove valuable for targeted therapy of pancreatic cancer.

Targeting heat‐shock protein 90 improves efficacy of rapamycin in a model of hepatocellular carcinoma in mice

Hepatocellular carcinoma (HCC) remains associated with a poor prognosis, but novel targeted therapies in combination with anti‐angiogenic substances may offer new perspectives. We hypothesized that simultaneous targeting of tumor cells, endothelial cells, and pericytes would reduce growth and angiogenesis of HCC, which represents a highly vascularized tumor entity. Recently, because of their anti‐angiogenic properties, inhibitors of mammalian target of rapamycin (mTOR) have entered clinical trials for therapy of HCC. However, treatment with mTOR inhibitors may lead to paradoxical activation of Akt signaling in tumor cells via insulin‐like growth factor‐I receptor (IGF‐IR)–dependent and IGF‐IR–independent mechanisms. Because we have recently identified heat shock protein 90 (Hsp90) antagonists to impair both oncogenic and angiogenic signaling cascades in tumor cells, including Akt and IGF‐IR, we sought to investigate whether Hsp90 blockade could improve growth‐inhibitory and anti‐angiogenic effects of the mTOR inhibitor rapamycin. Human HCC cells, a murine hepatoma cell line, endothelial cells (ECs), and vascular smooth muscle cells (VSMC) were employed in experiments. Results show that dual inhibition of mTOR and Hsp90 leads to effective disruption of oncogenic signaling cascades and substantially improves growth‐inhibitory effects in vivo. Importantly, blocking Hsp90 abrogated the rapamycin‐induced activation of Akt and of the downstream effector nuclear factor kappa‐B (NF‐κB) in HCC tumors. Furthermore, Hsp90 inhibition reduced the expression of platelet‐derived growth factor‐receptor‐β (PDGF‐Rβ) on VSMCs, and diminished vascular endothelial growth factor‐receptor 2 (VEGFR‐2) expression on ECs, which further improves the anti‐angiogenic capacity of this regimen. Conclusion: Blocking Hsp90 disrupts rapamycin‐induced activation of alternative signaling pathways in HCCs and substantially improves the growth‐inhibitory effects of mTOR inhibition in vivo. Hence, the concept of targeting tumor cells, ECs, and VSMCs by blocking Hsp90/mTOR could prove valuable for treatment of HCC. (HEPATOLOGY 2008.)

Mycophenolate mofetil inhibits tumor growth and angiogenesis in vitro but has variable antitumor effects in vivo, possibly related to bioavailability.

Background. Identifying immunosuppressive agents with antitumor effects could help address the problem of posttransplant malignancy. Here we tested for potential inhibitory effects of mycophenolate mofetil (MMF) on tumors in vitro and in vivo. Methods. Mouse CT26 colon adenocarcinoma, B16 melanoma, and human TMK1 gastric adenocarcinoma cells were tested for in vitro growth in the presence of MMF. In vitro angiogenesis was tested with a rat aortic-ring assay. Tumor cells were implanted into dorsal skinfold chambers (DSFC) to assess in vivo angiogenesis. Subcutaneous tumor growth was determined in mice receiving MMF. Results. MMF caused a dose-dependent reduction in tumor cell numbers in vitro, starting between 0.1 to 1 μM. Vessel sprouting from aortic rings was markedly blocked by similar concentrations of MMF. In vivo, however, DSFC results showed a marginal reduction in CT26 tumor angiogenesis with MMF doses of 40 or 80 mg/kg/day, although MMF did inhibit TMK1 vascularity. Moreover, 80 mg/kg/day MMF did not reduce subcutaneous CT26 tumor volumes, but did slightly inhibit B16 and TMK1 expansion. Interestingly, the mycophenolic acid (MPA) blood level 2 hr after 80 mg/kg/day MMF bolus dosing was near 14 mg/L, but decreased dramatically thereafter, suggesting a drug availability issue. Indeed, intermittent 2-hr MMF pulses in tumor-cell cultures substantially reduced the antiproliferative effect of MPA. Conclusion. MMF strongly inhibits tumor cell growth and angiogenesis in vitro, but only marginally inhibits tumors in vivo. These contrasting results may relate to drug availability, where intermittent exposure of tumor cells to immunosuppressive doses of MMF substantially reduce its potential antitumor effects.