Mice lacking functional CD95-ligand display reduced proliferation of the intestinal epithelium without gross homeostatic alterations.

Homeostasis of the continuously self-renewing intestinal tract involves cell proliferation, migration, differentiation along the crypt-villus-axis and shedding of cells into the gut lumen. CD95-ligand (FAS-ligand, CD95L) is a cytokine that is known for its capacity to induce apoptosis by binding its cognate receptor, CD95 (Fas). More recently, it was discovered that CD95L can also induce other cellular responses, such as proliferation, differentiation and cell migration. CD95L is highly expressed in Paneth cells of the small intestine which are in close contact with intestinal stem cells. This suggests a potential role for CD95L in controlling stem cell function and, possibly, intestinal homeostasis. We analyzed the intestines of mice deficient for functional CD95L (gld) for potential alterations in the diversity of stem-cell-lineages and parameters of intestinal homeostasis. Stem cell diversity was assessed by analyzing methylation patterns of the non-transcribed mMYOD gene. Proliferation was analyzed by BrdU labeling and differentiation was assessed by immunohistochemistry. Of all parameters analyzed, only epithelial cell proliferation was significantly reduced in the small intestines of gld-mice, but not in their colons which lack CD95L expression. We conclude that CD95L has a proliferation-stimulating role during normal turnover of the small intestine, but has a marginal effect on overall intestinal homeostasis.

GPx2 suppression of H2O2 stress links the formation of differentiated tumor mass to metastatic capacity in colorectal cancer.

Colorectal tumorigenesis is accompanied by the generation of oxidative stress, but how this controls tumor development is poorly understood. Here, we studied how the H2O2-reducing enzyme glutathione peroxidase 2 (GPx2) regulates H2O2 stress and differentiation in patient-derived "colonosphere" cultures. GPx2 silencing caused accumulation of radical oxygen species, sensitization to H2O2-induced apoptosis, and strongly reduced clone- and metastasis-forming capacity. Neutralization of radical oxygen species restored clonogenic capacity. Surprisingly, GPx2-suppressed cells also lacked differentiation potential and formed slow-growing undifferentiated tumors. GPx2 overexpression stimulated multilineage differentiation, proliferation, and tumor growth without reducing the tumor-initiating capacity. Finally, GPx2 expression was inversely correlated with H2O2-stress signatures in human colon tumor cohorts, but positively correlated with differentiation and proliferation. Moreover, high GPx2 expression was associated with early tumor recurrence, particularly in the recently identified aggressive subtype of human colon cancer. We conclude that H2O2 neutralization by GPx2 is essential for maintaining clonogenic and metastatic capacity, but also for the generation of differentiated proliferating tumor mass. The results reveal an unexpected redox-controlled link between tumor mass formation and metastatic capacity.

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.

The secretome of colon cancer stem cells contains drug-metabolizing enzymes.

Drug-resistant cancer stem cells (CSCs) have been implicated in tumor recurrence following chemotherapy. However, the contribution of CSCs to drug-resistance in colorectal cancer is unclear and CSC-intrinsic drug-resistance mechanisms are ill-defined. Here, we address these issues by proteomic analysis of the secretomes of CSCs and isogenic differentiated tumor cells (DTCs) isolated from three distinct metastasized colon tumors. Mass spectrometry-based proteomics identified 1254 unique proteins in the conditioned media of the paired CSC and DTC cultures. Ingenuity Pathway Analysis revealed that proteins governing 'Cell Death' were most significantly enriched in the CSC secretome. The vast majority of these (37/43) promote cell survival. The CSC secretome is also characterized by a pro-survival Nrf2 antioxidant signature. Interestingly, proteome-maintenance networks are highly enriched in the CSC secretome. CSCs also secrete high levels of drug-metabolizing enzymes, including aldehyde dehydrogenase 1 (ALDH1A1) and bleomycin hydrolase (BLMH). We show that these enzymes cause extracellular detoxification of maphosphamide and bleomycin respectively. We conclude that colorectal CSCs are characterized by extensive survival and anti-oxidant networks, which are likely to contribute to CSC-intrinsic drug-resistance. In addition, CSCs may modulate drug responses in nearby tumor cells by detoxifying chemotherapeutic drugs in the extracellular space. BIOLOGICAL SIGNIFICANCE: Cancer stem cells are thought to play an important role in mediating drug resistance and tumor recurrence following chemotherapy. Therefore, it is important to identify the factors that are secreted by them. Our results provide novel insights into the pathways that govern the intrinsic resistance of CSCs to chemotherapy and, furthermore, demonstrate that they can also inactivate chemotherapeutic drugs in the extracellular space. A better understanding of the pathways that govern drug resistance in CSCs may help in developing effective CSC-targeting drugs.

PDGFRB promotes liver metastasis formation of mesenchymal-like colorectal tumor cells.

In epithelial tumors, the platelet-derived growth factor receptor B (PDGFRB) is mainly expressed by stromal cells of mesenchymal origin. Tumor cells may also acquire PDGFRB expression following epithelial-to-mesenchymal transition (EMT), which occurs during metastasis formation. Little is known about PDGFRB signaling in colorectal tumor cells. We studied the relationship between PDGFRB expression, EMT, and metastasis in human colorectal cancer (CRC) cohorts by analysis of gene expression profiles. PDGFRB expression in primary CRC was correlated with short disease-free and overall survival. PDGFRB was co-expressed with genes involved in platelet activation, transforming growth factor beta (TGFB) signaling, and EMT in three CRC cohorts. PDGFRB was expressed in mesenchymal-like tumor cell lines in vitro and stimulated invasion and liver metastasis formation in mice. Platelets, a major source of PDGF, preferentially bound to tumor cells in a non-activated state. Platelet activation caused robust PDGFRB tyrosine phosphorylation on tumor cells in vitro and in liver sinusoids in vivo. Platelets also release TGFB, which is a potent inducer of EMT. Inhibition of TGFB signaling in tumor cells caused partial reversion of the mesenchymal phenotype and strongly reduced PDGFRB expression and PDGF-stimulated tumor cell invasion. These results suggest that PDGFRB may contribute to the aggressive phenotype of colorectal tumors with mesenchymal properties, most likely downstream of platelet activation and TGFB signaling.

Surgical implantation of an abdominal imaging window for intravital microscopy.

High-resolution intravital microscopy through imaging windows has become an indispensable technique for the long-term visualization of dynamic processes in living animals. Easily accessible sites such as the skin, the breast and the skull can be imaged using various different imaging windows; however, long-term imaging studies on cellular processes in abdominal organs are more challenging. These processes include colonization of the liver by metastatic tumor cells and the development of an immune response in the spleen. We have recently developed an abdominal imaging window (AIW) that allows long-term imaging of the liver, the pancreas, the intestine, the kidney and the spleen. Here we describe the detailed protocol for the optimal surgical implantation of the AIW, which takes ∼1 h, and subsequent multiphoton imaging, which takes up to 1 month.

Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis.

Cell dynamics in subcutaneous and breast tumors can be studied through conventional imaging windows with intravital microscopy. By contrast, visualization of the formation of metastasis has been hampered by the lack of long-term imaging windows for metastasis-prone organs, such as the liver. We developed an abdominal imaging window (AIW) to visualize distinct biological processes in the spleen, kidney, small intestine, pancreas, and liver. The AIW can be used to visualize processes for up to 1 month, as we demonstrate with islet cell transplantation. Furthermore, we have used the AIW to image the single steps of metastasis formation in the liver over the course of 14 days. We observed that single extravasated tumor cells proliferated to form "pre-micrometastases," in which cells lacked contact with neighboring tumor cells and were active and motile within the confined region of the growing clone. The clones then condensed into micrometastases where cell migration was strongly diminished but proliferation continued. Moreover, the metastatic load was reduced by suppressing tumor cell migration in the pre-micrometastases. We suggest that tumor cell migration within pre-micrometastases is a contributing step that can be targeted therapeutically during liver metastasis formation.

CD95 signaling in colorectal cancer.

CD95 and its ligand (CD95L) are widely expressed in colorectal tumors, but their role in shaping tumor behavior is unclear. CD95 activation on tumor cells can lead to apoptosis, while CD95L attracts neutrophils, suggesting a function in tumor suppression. However, CD95 can also promote tumorigenesis, at least in part by activating non-apoptotic signaling pathways that stimulate tumor cell proliferation, invasion and survival. In addition, CD95 signaling in stromal cells and tumor-infiltrating inflammatory cells has to be taken into account when addressing the function of CD95 and its ligand in colorectal tumor biology. We present a model in which the tumor-suppressing and tumor-promoting activities of CD95/CD95L together determine colorectal tumor behavior. We also discuss how these multiple activities are changing our view of CD95 and CD95L as potential therapeutic targets in the treatment of colorectal cancer. We conclude that locking CD95 in apoptosis-mode may be a more promising anti-cancer strategy than simply inhibiting or stimulating CD95.

Studying primary tumor-associated fibroblast involvement in cancer metastasis in mice.

Stromal cells have been studied extensively in the primary tumor microenvironment. In addition, mesenchymal stromal cells may participate in several steps of the metastatic cascade. Studying this interaction requires methods to distinguish and target stromal cells originating from the primary tumor versus their counterparts in the metastatic site. Here we illustrate a model of human tumor stromal cell-mouse cancer cell coimplantation. This model can be used to selectively deplete human stromal cells (using diphtheria toxin, DT) without affecting mouse cancer cells or host-derived stromal cells. Establishment of novel genetic models (e.g., transgenic expression of the DT receptor in specific cells) may eventually allow analogous models using syngeneic cells. Studying the role of stromal cells in metastasis using the model outlined above may take 8 weeks.

How CD95 stimulates invasion.

CD95 is best known for its capacity to induce apoptosis, but also activates multiple non-apoptotic signalling pathways. In particular, CD95 promotes migration and tissue invasion of apoptosis-resistant cell types, and this plays a central role in inflammation, neurobiology, and tumor biology. CD95 induces invasion by stimulating the expression of extracellular matrix (ECM)-degrading proteases, and by stimulating the formation of actin-driven cell protrusions through Rac and the cofilin pathway. In this review we discuss how CD95-initiated signalling pathways may cooperate to facilitate cell migration and tissue invasion.

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-ß, resulting in ligand-stimulated phosphatidylinositol (4,5)-bisphosphate (PIP(2)) hydrolysis. PIP(2) 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.

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.

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

BACKGROUND: 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. METHODS: Mice underwent vascular clamping 5 days after induction of colorectal liver metastases. Invasion and outgrowth of micrometastases following I/R were analyzed by post-mortem confocal microscopy (36 hr post-I/R) and by morphometric assessment of tumor load (5 days post-I/R), respectively. Tumor cell CD95 was suppressed by lentiviral RNA interference. The contribution of host CD95L was assessed by using gld-mice lacking functional CD95L. RESULTS: CD95-knockdown in tumor cells strongly reduced perinecrotic invasion (tumor diameter from ∼830 to ∼470 µm) and largely prevented outgrowth acceleration of perinecrotic liver metastases following I/R (from ∼8- to ∼4.5-fold). In gld-mice, the relative hepatic area with necrosis was markedly reduced. Perinecrotic tumor cell clusters still displayed an invasive phenotype (tumor diameter of ∼980 µm). However, I/R-induced acceleration of tumor outgrowth was reduced in gld-mice from ∼8- to ∼5-fold. CONCLUSIONS: I/R induces invasion and accelerated outgrowth of preestablished metastases in a CD95-dependent manner. Activation of the CD95 system following I/R not only contributes to liver injury, but may also promote aggressive tumor recurrence.

Synergistic killing of colorectal cancer cells by oxaliplatin and ABT-737.

BACKGROUND: Oxaliplatin is frequently used in the treatment of metastatic colorectal cancer (CRC). Our previous work shows that oxaliplatin induces the pro-apoptotic protein Noxa in CRC cells. The Bcl2-inhibitor ABT-737 is particularly effective in cells with high Noxa levels. Therefore, we tested whether oxaliplatin and ABT-737 display synergy in killing CRC cells. METHODS: A panel of CRC cell lines was treated with oxaliplatin and ABT-737, either alone or in combination. Apoptosis was measured by FACS analysis of sub-G1 DNA content and by Western blot analysis of caspase-3 processing. Noxa expression was suppressed by lentiviral RNA interference. RESULTS: Oxaliplatin and ABT-737 displayed a strong synergistic apoptotic response, which was dependent on wildtype TP53 and oncogenic KRAS. TP53 and KRAS were required for drug-induced Noxa expression and this was essential for tumor cell apoptosis. Oxaliplatin, but not ABT-737, induced p53 accumulation, but both drugs stimulated Noxa expression. Combination treatment of mice with subcutaneous tumor xenografts drastically reduced tumor volume, while single drug treatment had no effect. CONCLUSION: ABT-737 synergizes with oxaliplatin to kill colorectal cancer cells. This requires induction of Noxa by wildtype TP53 and oncogenic KRAS. Future studies should explore the anti-tumor efficacy of this drug combination in mouse models for spontaneous CRC development and in patient-derived tumor cell cultures and xenografts.

Malignant cells facilitate lung metastasis by bringing their own soil.

Metastatic cancer cells (seeds) preferentially grow in the secondary sites with a permissive microenvironment (soil). We show that the metastatic cells can bring their own soil--stromal components including activated fibroblasts--from the primary site to the lungs. By analyzing the efferent blood from tumors, we found that viability of circulating metastatic cancer cells is higher if they are incorporated in heterotypic tumor-stroma cell fragments. Moreover, we show that these cotraveling stromal cells provide an early growth advantage to the accompanying metastatic cancer cells in the lungs. Consistent with this hypothesis, we demonstrate that partial depletion of the carcinoma-associated fibroblasts, which spontaneously spread to the lung tissue along with metastatic cancer cells, significantly decreases the number of metastases and extends survival after primary tumor resection. Finally, we show that the brain metastases from lung carcinoma and other carcinomas in patients contain carcinoma-associated fibroblasts, in contrast to primary brain tumors or normal brain tissue. Demonstration of the direct involvement of primary tumor stroma in metastasis has important conceptual and clinical implications for the colonization step in tumor progression.

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.

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.