Klaas M. Govaert

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.

The death receptor CD95 activates the cofilin pathway to stimulate tumour cell invasion

The death receptor CD95 promotes apoptosis through well‐defined signalling pathways. In colorectal cancer cells, CD95 primarily stimulates migration and invasion through pathways that are incompletely understood. Here, we identify a new CD95‐activated tyrosine kinase pathway that is essential for CD95‐stimulated tumour cell invasion. We show that CD95 promotes Tyr 783 phosphorylation of phospholipase C‐γ1 through the platelet‐derived growth factor receptor‐β, resulting in ligand‐stimulated phosphatidylinositol (4,5)‐bisphosphate (PIP2) hydrolysis. PIP2 hydrolysis liberates the actin‐severing protein cofilin from the plasma membrane to initiate cortical actin remodelling. Cofilin activation is required for CD95‐stimulated formation of membrane protrusions and increased tumour cell invasion.

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).

Hypoxia After Liver Surgery Imposes an Aggressive Cancer Stem Cell Phenotype on Residual Tumor Cells

Objective:To assess the contribution of hypoxia and bone marrow–derived cells to aggressive outgrowth of micrometastases after liver surgery. Background:Liver surgery generates a microenvironment that fosters aggressive tumor recurrence. These areas are characterized by chronic hypoxia and influx of bone marrow–derived cells. Methods:The contribution of hematopoietic cell types was studied in mice lacking specific components of the immune system and in irradiated mice lacking all bone marrow–derived cells. Tumor cells were derived from colorectal cancer patients and from a metastatic tumor cell line. Hypoxia-induced changes in stem cell and differentiation marker expression, clone-forming potential, and metastatic capacity were assessed. The effect of vascular clamping on cancer stem cell (CSC) characteristics was performed in mice bearing patient-derived liver metastases. Results:Immune cells and bone marrow–derived cells were not required for aggressive outgrowth of micrometastases in livers treated with surgery. Rather, hypoxia was sufficient to promote invasion and accelerate metastatic outgrowth. This was associated with a rapid loss of differentiation markers and increased expression of CSC markers and clone-forming capacity. Likewise, metastases residing in ischemia-reperfusion-injured liver lobes acquired CSC characteristics. Despite their renowned general resistance to chemotherapy, clone-forming CSCs were readily killed by the hypoxia-activated prodrug tirapazamine. Conclusions:Surgery-generated hypoxia in the liver causes rapid dedifferentiation of tumor cells into immature CSCs with high clone- and metastasis-forming capacity. The results help explain the phenomenon of aggressive local tumor recurrence after liver surgery and offer a potential strategy to kill aggressive CSCs by hypoxia-activated prodrugs.

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.

Maintenance of Clonogenic KIT+ Human Colon Tumor Cells Requires Secretion of Stem Cell Factor by Differentiated Tumor Cells

BACKGROUND & AIMS Colon tumors contain a fraction of undifferentiated stem cell-like cancer cells with high tumorigenic potential. Little is known about the signals that maintain these stem-like cells. We investigated whether differentiated tumor cells provide support. METHODS We established undifferentiated colonosphere cultures from human colon tumors and used them to generate stably differentiated cell lines. Antibody arrays were used to identify secreted factors. Expression of genes involved in stemness, differentiation, and the epithelial to mesenchymal transition was measured using reverse transcription quantitative polymerase chain reaction. Expression of KIT in human tumors was analyzed with gene expression arrays and by immunohistochemistry. Colonospheres were injected into the livers of CBy.Cg-Foxn1nu/J mice. After liver tumors had formed, hypoxia was induced by vascular clamping. RESULTS Differentiated cells from various tumors, or medium conditioned by them, increased the clonogenic capacity of colonospheres. Stem cell factor (SCF) was secreted by differentiated tumor cells and supported the clonogenic capacity of KIT(+) colonosphere cells. Differentiated tumor cells induced the epithelial to mesenchymal transition in colonosperes; this was prevented by inhibition of KIT or SCF. SCF prevented loss of clonogenic potential under differentiation-inducing conditions. Suppression of SCF or KIT signaling greatly reduced the expression of genes that regulate stemness and the epithelial to mesenchymal transition and inhibited clonogenicity and tumor initiation. Bioinformatic and immunohistochemical analyses revealed a correlation between expression of KIT- and hypoxia-related genes in colon tumors, which was highest in relapse-prone mesenchymal-type tumors. Hypoxia induced expression of KIT in cultured cells and in human colon tumor xenografts and this contributed to the clonogenic capacity of the tumor cells. CONCLUSIONS Paracrine signaling from SCF to KIT, between differentiated tumor cells and undifferentiated stem-like tumor cells, helps maintain the stem-like features of tumor cells, predominantly under conditions of hypoxia.

Does Radiofrequency Ablation Add to Chemotherapy for Unresectable Liver Metastases

In patients with unresectable colorectal liver metastases (CRLM), radiofrequency ablation (RFA) might be a good alternative, whenever possible. In contrast to systemic therapy, the aim of RFA is to achieve complete local tumor control in an attempt to provide long-term survival. In this article we discuss the available evidence regarding the treatment of patients with unresectable CRLM, focusing on RFA in conjunction with modern systemic therapies. We observed that the available evidence in the existing literature is limited, and often consists of level 2 and 3 evidence, thereby hampering any firm conclusions. Nonetheless, RFA seems superior to chemotherapy alone in patients with liver-only disease amenable for RFA. However, the combination of RFA and chemotherapy has been demonstrated to be feasible and safe, lending support to the concept of RFA followed by chemotherapy, in order to reduce local recurrence rates and prolong survival.

A role for CD95 signaling in ischemia/reperfusion‐induced invasion and outgrowth of colorectal micrometastases in mouse liver

Ischemia/reperfusion (I/R) injury in the liver is associated with accelerated outgrowth of micrometastases. The aim of the study was to test the role of CD95 signaling in accelerated outgrowth of colorectal liver metastases following I/R.

Neoadjuvant chemotherapy affects molecular classification of colorectal tumors

The recent discovery of ‘molecular subtypes’ in human primary colorectal cancer has revealed correlations between subtype, propensity to metastasize and response to therapy. It is currently not known whether the molecular tumor subtype is maintained after distant spread. If this is the case, molecular subtyping of the primary tumor could guide subtype-targeted therapy of metastatic disease. In this study, we classified paired samples of primary colorectal carcinomas and their corresponding liver metastases (n=129) as epithelial-like or mesenchymal-like, using a recently developed immunohistochemistry-based classification tool. We observed considerable discordance (45%) in the classification of primary tumors and their liver metastases. Discordant classification was significantly associated with the use of neoadjuvant chemotherapy. Furthermore, gene expression analysis of chemotherapy-exposed versus chemotherapy naive liver metastases revealed expression of a mesenchymal program in pre-treated tumors. To explore whether chemotherapy could cause gene expression changes influencing molecular subtyping, we exposed patient-derived colonospheres to six short cycles of 5-fluorouracil. Gene expression profiling and signature enrichment analysis subsequently revealed that the expression of signatures identifying mesenchymal-like tumors was strongly increased in chemotherapy-exposed tumor cultures. Unsupervised clustering of large cohorts of human colon tumors with the chemotherapy-induced gene expression program identified a poor prognosis mesenchymal-like subgroup. We conclude that neoadjuvant chemotherapy induces a mesenchymal phenotype in residual tumor cells and that this may influence the molecular classification of colorectal tumors.

Effects of tirapazamine on experimental colorectal liver metastases after radiofrequency ablation

Radiofrequency ablation (RFA) is a common procedure for the management of colorectal liver metastases. RFA‐generated lesions are surrounded by a rim of hypoxia that is associated with aggressive outgrowth of intrahepatic micrometastases. Hypoxia‐activated prodrugs such as tirapazamine are designed selectively to induce apoptosis in tumour cells under hypoxic conditions. Therefore, it was hypothesized that tirapazamine may have therapeutic value in limiting hypoxia‐associated tumour outgrowth following RFA.