Maarten W. Nijkamp

Oncogenic K-Ras Turns Death Receptors Into Metastasis-Promoting Receptors in Human and Mouse Colorectal Cancer Cells

BACKGROUND & AIMS Death receptors expressed on tumor cells can prevent metastasis formation by inducing apoptosis, but they also can promote migration and invasion. The determinants of death receptor signaling output are poorly defined. Here we investigated the role of oncogenic K-Ras in determining death receptor function and metastatic potential. METHODS Isogenic human and mouse colorectal cancer cell lines differing only in the presence or absence of the K-Ras oncogene were tested in apoptosis and invasion assays using CD95 ligand and tumor necrois factor-related apoptosis-inducing ligand (TRAIL) as stimuli. Metastatic potential was assessed by intrasplenic injections of green fluorescent protein- or luciferase-expressing tumor cells, followed by intravital fluorescence microscopy or bioluminescence imaging, and confocal microscopy and immunohistochemistry. Ras-effector pathway control of CD95 output was assessed by an RNA-interference and inhibitor-based approach. RESULTS CD95 ligand and TRAIL stimulated invasion of colorectal tumor cells and liver metastases in a K-Ras-dependent fashion. Loss of mutant K-Ras switched CD95 and TRAIL receptors back into apoptosis mode and abrogated metastatic potential. Raf1 was essential for the switch in CD95 function, for tumor cell survival in the liver, and for K-Ras-driven formation of liver metastases. K-Ras and Raf1 suppressed Rho kinase (ROCK)/LIM kinase-mediated phosphorylation of the actin-severing protein cofilin. Overexpression of ROCK or LIM kinase allowed CD95L to induce apoptosis in K-Ras-proficient cells and prevented metastasis formation, whereas their suppression protected K-Ras-deficient cells against apoptosis. CONCLUSIONS Oncogenic K-Ras and its effector Raf1 convert death receptors into invasion-inducing receptors by suppressing the ROCK/LIM kinase pathway, and this is essential for K-Ras/Raf1-driven metastasis formation.

Differentiated Human Colorectal Cancer Cells Protect Tumor-Initiating Cells From Irinotecan

BACKGROUND & AIMS Stem cells of normal tissues have resistance mechanisms that allow them to survive genotoxic insults. The stem cell-like cells of tumors are defined by their tumor-initiating capacity and may have retained these resistance mechanisms, making them resistant to chemotherapy. We studied the relationship between resistance to the topoisomerase I inhibitor irinotecan and tumor-initiating potential in human colonosphere cultures and in mice with colorectal xenograft tumors. METHODS Colonosphere cultures were established from human colorectal tumor specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. Stem cell and differentiation markers were analyzed by immunoblotting and fluorescence-activated cell sorting. Clone- and tumor-initiating capacities were assessed by single-cell cloning and in immune-deficient mice. Sensitivity to irinotecan was assessed in vitro and in tumor-bearing mice. The relationship between drug resistance and tumor-initiating capacity was tested by fluorescence-activated cell sorting of colonosphere cells, based on expression of ABCB1 and aldehyde dehydrogenase (ALDH) activity. RESULTS Colonosphere cultures had a high capacity to initiate tumors in mice and were resistant to irinotecan. Inhibition of the drug-efflux pump ABCB1 by PSC-833 allowed irinotecan to eradicate tumor-initiating cells. However, ABCB1 was expressed only by a subpopulation of differentiated tumor cells that did not form clones or tumors. Conversely, tumor-initiating cells were ABCB1-negative and were identified by high ALDH activity. Tumorigenic ALDHhigh/ABCB1negative cells generated nontumorigenic ALDHlow/ABCB1positive daughter cells in vitro and in tumor xenografts. PSC-833 increased the antitumor efficacy of irinotecan in mice. CONCLUSIONS The resistance of colorectal tumors to irinotecan requires the cooperative action of tumor-initiating ALDHhigh/ABCB1negative cells and their differentiated, drug-expelling, ALDHlow/ABCB1positive daughter cells.

Accelerated perinecrotic outgrowth of colorectal liver metastases following radiofrequency ablation is a hypoxia-driven phenomenon.

Objective:The aim of this study was to assess how thermal ablation of colorectal liver metastases affects the outgrowth of micrometastases in the transition zone (TZ) between ablated tissue and the unaffected reference zone (RZ) in 2 different murine models. Background:Thermal destruction therapies of nonresectable colorectal liver metastases, including radiofrequency ablation (RFA), can provide tumor clearance, but local recurrences are common. Methods:Three days after intrasplenic injection of C26 colon carcinoma cells, RFA was applied to the left liver lobe. Perinecrotic microcirculation, tissue hypoxia, hypoxia inducible factor (HIF)-1α and HIF-2α, and the outgrowth of micrometastases both in the TZ and in the RZ were evaluated over time. Results:In 2 different animal models, the outgrowth of micrometastases in the TZ following RFA was stimulated approximately 4-fold compared to tumor growth in the RZ. Accelerated tumor growth in the TZ was associated with microcirculatory disturbances, prolonged hypoxia, and stabilization of HIF-1α and HIF-2α in the tumor cells. In addition, RFA induced the formation of new hepatic vessels that sprouted from existing sinusoids and grew into the generated necrotic lesion. Surprisingly, the accelerated tumor growth was not associated with these vessels. Treatment with 17DMAG prevented HIF-1α and HIF-2α stabilization and selectively reduced tumor growth in the TZ by ∼40% without affecting tumor growth in sham-operated mice or in the RZ of RFA-treated mice. PTK787/ZK-222584, a nonselective Vascular Endothelial Growth Factor (VEGF)-receptor inhibitor, reduced RFA-stimulated tumor growth and tumor growth in the RZ to a similar extent. Conclusions:We conclude that RFA stimulates the outgrowth of tumor cells at the lesion periphery. Angiogenesis is not the driving force behind RFA-stimulated tumor growth, but other hypoxia/HIF-activated pathways are likely to be important.

CD95 is a key mediator of invasion and accelerated outgrowth of mouse colorectal liver metastases following radiofrequency ablation

BACKGROUND & AIMS Recently, we have shown that micro-metastases, in the hypoxic transition zone surrounding lesions generated by radiofrequency ablation (RFA), display strongly accelerated outgrowth. CD95 is best known for its ability to induce apoptosis but can also promote tumorigenesis in apoptosis-resistant tumor cells. Therefore, we tested whether CD95 signaling plays a role in accelerated outgrowth of colorectal liver metastases following RFA. METHODS Hypoxia-induced invasion was assessed in three-dimensional EGFP-expressing C26 tumor cell cultures by confocal microscopy. CD95 localization was tested by immunofluorescence. Invasion and outgrowth of liver metastases following RFA were analyzed by post-mortem confocal microscopy and by morphometric assessment of tumor load. Neutralization of CD95L was performed by using antibody MFL4. CD95 was suppressed by lentiviral RNA interference. The role of host CD95L was assessed using gld mice. RESULTS Micro-metastases in the hypoxic transition zone following RFA displayed a highly invasive phenotype and increased expression of CD95 and CD95L. Hypoxia-induced tumor cell invasion in vitro increased the expression of CD95 and CD95L and induced translocation of CD95 to the invasive front. In vitro invasion, metastasis invasion, and accelerated tumor growth in the transition zone were strongly suppressed by neutralizing CD95L or by suppressing tumor cell CD95. In contrast, metastasis invasion and outgrowth were unaffected in gld mice. CONCLUSIONS Hypoxia causes autocrine activation of CD95 on colorectal tumor cells, thereby promoting local invasion and accelerated metastasis outgrowth in the hypoxic transition zone following RFA. Further pre-clinical work is needed to assess the role of CD95L neutralization, either alone or in combination with chemotherapy, in limiting aggressive recurrence of liver metastases following RFA.

Prolonged portal triad clamping during liver surgery for colorectal liver metastases is associated with decreased time to hepatic tumour recurrence

AIMS The aim of this study was to evaluate the oncological outcome of portal triad clamping during hepatectomy in colorectal cancer patients. METHODS 160 patients with colorectal liver metastases underwent a partial hepatectomy with curative intent. Data were collected in a prospective database and were retrospectively analyzed for time to liver recurrence (TTLiR) and time to overall recurrence (TTR). The prognostic significance of portal triad clamping of any type and severe ischemia due to prolonged portal triad clamping was determined by Cox regression models. RESULTS TTLiR was reduced after clamping of any type, although not statistically significant (p=0.061). Severe ischemia due to prolonged portal triad clamping significantly decreased TTLiR (p=0.022), but not TTR. Furthermore, severe ischemia independently predicted TTLiR in a multivariable analysis (p=0.038). CONCLUSIONS Severe ischemia due to prolonged portal triad clamping during hepatic resection for colorectal liver metastases appears to be associated with decreased TTLiR. Further research remains necessary to determine the causative effect of prolonged vascular clamping on liver tumour recurrence.

Incomplete thermal ablation stimulates proliferation of residual renal carcinoma cells in a translational murine model

Whats known on the subject? and What does the study add?

Radiofrequency ablation of colorectal liver metastases induces an inflammatory response in distant hepatic metastases but not in local accelerated outgrowth.

Recently, we have shown in a murine model that radiofrequency ablation (RFA) induces accelerated outgrowth of colorectal micrometastases in the transition zone (TZ) surrounding the ablated lesion. Conversely, RFA also induces an anti‐tumor T‐cell response that may limit tumor growth at distant sites. Here we have evaluated whether an altered density of inflammatory cells could be observed in the perinecrotic (TZ) metastases compared to hepatic metastases in the distant reference zone (RZ).

Liver surgery induces an immediate mobilization of progenitor cells in liver cancer patients: A potential role for G-CSF.

Background: In preclinical models recruitment of bone-marrow derived (endothelial) progenitor cells (BD(E)PCs) contributes to tumor growth and metastasis formation. Here we investigated whether these (E)PCs and mobilizing cytokines are released after partial hepatectomy or radiofrequency ablation (RFA) for liver tumors. In addition, we tested whether G-CSF could play a role in EPC mobilization in mice and in human volunteers. Methods: Before, during and after liver surgery plasma and mononuclear cells were collected from 12 patients undergoing partial hepatectomy or RFA. To explore the role of G-CSF C57Bl/6 mice and 20 human volunteers received G-CSF (0.3 or 3 μg). In all individuals, (E)PC numbers were determined by flow cytometry at predefined timepoints shortly after therapy. Plasma levels of G-CSF, VEGF and SDF-1α were measured by ELISA> Results: Patients undergoing partial hepatectomy or RFA showed a instantaneous release of EPCs following laparotomy and mobilization of the liver. Elevated EPC levels were maintained during the entire procedure, but dropped to near-baseline levels 4 hours after completion of the procedure. Plasma G-CSF levels showed a 5-10-fold increase after the procedure and low-dose G-CSF administration to mice or healthy volunteers was sufficient to induce an immediate release of EPCs. Surgery also caused an increase in the plasma levels of VEGF, but not SDF1. Conclusion: Compliant with previous published data concerning VDA and chemotherapy treatment,liver surgery induces an instantaneous release of EPCs, conceivably in response to elevated G-CSF levels. This suggests the value of exploring therapeutic avenues to prevent this process.

Multipolar radiofrequency ablation for colorectal liver metastases close to major hepatic vessels

BACKGROUND Resection of colorectal liver metastases (CRLM) is often hindered by their location close to the major hepatic vessels. So far, radiofrequency ablation for perivascular tumours was thought to be ineffective and unsafe due to either the heat sink effect or vascular thrombosis. The aim of this study was to examine whether RFA using multipolar probes could be a safe and effective option for CRLM adjacent to major hepatic vessels. METHODS Patients were treated with multipolar RFA during an open procedure using 3 simultaneously placed electrodes. In 52 consecutive patients with CRLM, 144 tumours were ablated with RFA. In 16 out of 52 (31%) patients, metastases were abutting major hepatic vessels. We examined whether perivascular location was a risk factor for local tumour progression. The relation between perivascular location and time to local tumour progression and recurrence free survival was assessed using cox-regression analysis. RESULTS All patients were followed for at least 3 years after RFA unless they deceased before this time. Local tumour progression following RFA occurred in 17 out of 144 tumours (12%), of which 4 out of 21 were perivascular tumours. Tumour size was the only risk factor for local tumour progression in this study. Proximity to large vessels was neither a risk factor for local local tumour progression, nor for time to local tumour progression or recurrence free survival. DISCUSSION This study indicates that patients with CRLM abutting any of the large hepatic vessels can be safe and effectively treated with RFA when using a multipolar system.

Exploring gene expression signatures for predicting disease free survival after resection of colorectal cancer liver metastases.

Background and Objectives This study was designed to identify and validate gene signatures that can predict disease free survival (DFS) in patients undergoing a radical resection for their colorectal liver metastases (CRLM). Methods Tumor gene expression profiles were collected from 119 patients undergoing surgery for their CRLM in the Paul Brousse Hospital (France) and the University Medical Center Utrecht (The Netherlands). Patients were divided into high and low risk groups. A randomly selected training set was used to find predictive gene signatures. The ability of these gene signatures to predict DFS was tested in an independent validation set comprising the remaining patients. Furthermore, 5 known clinical risk scores were tested in our complete patient cohort. Result No gene signature was found that significantly predicted DFS in the validation set. In contrast, three out of five clinical risk scores were able to predict DFS in our patient cohort. Conclusions No gene signature was found that could predict DFS in patients undergoing CRLM resection. Three out of five clinical risk scores were able to predict DFS in our patient cohort. These results emphasize the need for validating risk scores in independent patient groups and suggest improved designs for future studies.