High-throughput confocal imaging of differentiated 3D liver-like spheroid cellular stress response reporters for identification of drug-induced liver injury liability.

Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult. Here, we applied our bacterial artificial chromosome-based GFP reporter cell lines representing Nrf2 activation (Srxn1-GFP and NQO1-GFP), unfolded protein response (BiP-GFP and Chop-GFP), and DNA damage response (p21-GFP and Btg2-GFP) as long-term differentiated 3D liver-like spheroid cultures. All HepG2 GFP reporter lines differentiated into 3D spheroids similar to wild-type HepG2 cells. We systematically optimized the automated imaging and quantification of GFP reporter activity in individual spheroids using high-throughput confocal microscopy with a reference set of DILI compounds that activate these three stress response pathways at the transcriptional level in primary human hepatocytes. A panel of 33 compounds with established DILI liability was further tested in these six 3D GFP reporters in single 48 h treatment or 6 day daily repeated treatment. Strongest stress response activation was observed after 6-day repeated treatment, with the BiP and Srxn1-GFP reporters being most responsive and identified particular severe-DILI-onset compounds. Compounds that showed no GFP reporter activation in two-dimensional (2D) monolayer demonstrated GFP reporter stress response activation in 3D spheroids. Our data indicate that the application of BAC-GFP HepG2 cellular stress reporters in differentiated 3D spheroids is a promising strategy for mechanism-based identification of compounds with liability for DILI.

Activation of the Nrf2 response by intrinsic hepatotoxic drugs correlates with suppression of NF-κB activation and sensitizes toward TNFα-induced cytotoxicity.

Drug-induced liver injury (DILI) is an important problem both in the clinic and in the development of new safer medicines. Two pivotal adaptation and survival responses to adverse drug reactions are oxidative stress and cytokine signaling based on the activation of the transcription factors Nrf2 and NF-κB, respectively. Here, we systematically investigated Nrf2 and NF-κB signaling upon DILI-related drug exposure. Transcriptomics analyses of 90 DILI compounds in primary human hepatocytes revealed that a strong Nrf2 activation is associated with a suppression of endogenous NF-κB activity. These responses were translated into quantitative high-content live-cell imaging of induction of a selective Nrf2 target, GFP-tagged Srxn1, and the altered nuclear translocation dynamics of a subunit of NF-κB, GFP-tagged p65, upon TNFR signaling induced by TNFα using HepG2 cells. Strong activation of GFP-Srxn1 expression by DILI compounds typically correlated with suppression of NF-κB nuclear translocation, yet reversely, activation of NF-κB by TNFα did not affect the Nrf2 response. DILI compounds that provided strong Nrf2 activation, including diclofenac, carbamazepine and ketoconazole, sensitized toward TNFα-mediated cytotoxicity. This was related to an adaptive primary protective response of Nrf2, since loss of Nrf2 enhanced this cytotoxic synergy with TNFα, while KEAP1 downregulation was cytoprotective. These data indicate that both Nrf2 and NF-κB signaling may be pivotal in the regulation of DILI. We propose that the NF-κB-inhibiting effects that coincide with a strong Nrf2 stress response likely sensitize liver cells to pro-apoptotic signaling cascades induced by intrinsic cytotoxic pro-inflammatory cytokines.

The residence time of focal adhesion kinase (FAK) and paxillin at focal adhesions in renal epithelial cells is determined by adhesion size, strength and life cycle status.

Focal adhesions (FAs) are specialized membrane-associated multi-protein complexes that link the cell to the extracellular matrix and enable cell proliferation, survival and motility. Despite the extensive description of the molecular composition of FAs, the complex regulation of FA dynamics is unclear. We have used photobleaching assays of whole cells to determine the protein dynamics in every single focal adhesion. We identified that the focal adhesion proteins FAK and paxillin exist in two different states: a diffuse cytoplasmic pool and a transiently immobile FA-bound fraction with variable residence times. Interestingly, the average residence time of both proteins increased with focal adhesion size. Moreover, increasing integrin clustering by modulating surface collagen density increased residence time of FAK but not paxillin. Finally, this approach was applied to measure FAK and paxillin dynamics using nocodazole treatment followed by washout. This revealed an opposite residence time of FAK and paxillin in maturing and disassembling FAs, which depends on the ventral and peripheral cellular position of the FAs.

Diclofenac inhibits tumor necrosis factor-α-induced nuclear factor-κB activation causing synergistic hepatocyte apoptosis.

UNLABELLED: Drug-induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor α (TNF-α). HepG2 cells were treated with diclofenac followed by TNF-α challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF-α. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF-α-mediated enhancement involved activation of caspase-3 through a caspase-8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c-Jun N-terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF-α-induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF-α-mediated nuclear factor kappaB (NF-κB) translocation oscillation in association with reduced NF-κB transcriptional activity. This was associated with inhibition by diclofenac of the TNF-α-induced phosphorylation of the inhibitor of NF-κB alpha (IκBα). Finally, inhibition of IκB kinase ß (IKKß) with BMS-345541 as well as stable lentiviral short hairpin RNA (shRNA)-based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNF-α-induced cytotoxicity. CONCLUSION: Together, our data suggest a model whereby diclofenac-mediated stress signaling suppresses TNF-α-induced survival signaling routes and sensitizes cells to apoptosis.

Elevated insulin-like growth factor 1 receptor signaling induces antiestrogen resistance through the MAPK/ERK and PI3K/Akt signaling routes.

INTRODUCTION: Insulin-like growth factor 1 (IGF-1) receptor (IGF-1R) is phosphorylated in all breast cancer subtypes. Past findings have shown that IGF-1R mediates antiestrogen resistance through cross-talk with estrogen receptor (ER) signaling and via its action upstream of the epidermal growth factor receptor and human epidermal growth factor receptor 2. Yet, the direct role of IGF-1R signaling itself in antiestrogen resistance remains obscure. In the present study, we sought to elucidate whether antiestrogen resistance is induced directly by IGF-1R signaling in response to its ligand IGF-1 stimulation. METHODS: A breast cancer cell line ectopically expressing human wild-type IGF-1R, MCF7/IGF-1R, was established by retroviral transduction and colony selection. Cellular antiestrogen sensitivity was evaluated under estrogen-depleted two-dimensional (2D) and 3D culture conditions. Functional activities of the key IGF-1R signaling components in antiestrogen resistance were assessed by specific kinase inhibitor compounds and small interfering RNA. RESULTS: Ectopic expression of IGF-1R in ER-positive MCF7 human breast cancer cells enhanced IGF-1R tyrosine kinase signaling in response to IGF-1 ligand stimulation. The elevated IGF-1R signaling rendered MCF7/IGF-1R cells highly resistant to the antiestrogens tamoxifen and fulvestrant. This antiestrogen-resistant phenotype involved mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase/protein kinase B pathways downstream of the IGF-1R signaling hub and was independent of ER signaling. Intriguingly, a MAPK/ERK-dependent agonistic behavior of tamoxifen at low doses was triggered in the presence of IGF-1, showing a mild promitogenic effect and increasing ER transcriptional activity. CONCLUSIONS: Our data provide evidence that the IGF-1/IGF-1R signaling axis may play a causal role in antiestrogen resistance of breast cancer cells, despite continuous suppression of ER transcriptional function by antiestrogens.

Systems microscopy approaches to understand cancer cell migration and metastasis.

Cell migration is essential in a number of processes, including wound healing, angiogenesis and cancer metastasis. Especially, invasion of cancer cells in the surrounding tissue is a crucial step that requires increased cell motility. Cell migration is a well-orchestrated process that involves the continuous formation and disassembly of matrix adhesions. Those structural anchor points interact with the extra-cellular matrix and also participate in adhesion-dependent signalling. Although these processes are essential for cancer metastasis, little is known about the molecular mechanisms that regulate adhesion dynamics during tumour cell migration. In this review, we provide an overview of recent advanced imaging strategies together with quantitative image analysis that can be implemented to understand the dynamics of matrix adhesions and its molecular components in relation to tumour cell migration. This dynamic cell imaging together with multiparametric image analysis will help in understanding the molecular mechanisms that define cancer cell migration.

Complete focal adhesion kinase deficiency in the mammary gland causes ductal dilation and aberrant branching morphogenesis through defects in Rho kinase-dependent cell contractility.

The adult, virgin mammary gland is a highly organized branched ductal network comprising two major cell types: myoepithelial and luminal epithelial cells. To study the role and mechanism of focal adhesion kinase (FAK)-mediated signaling in mammary gland development and differentiation, we used a conditional Fak-knockout mammary epithelial cell (MEC) transplantation model. Conditional Cre recombinase (Cre)-mediated Fak deletion in primary cultured MECs isolated from FAK(lox/lox)/Rosa26Cre-ERT2 donor mice caused loss of FAK in all mammary cells. Transplantation of Fak-knockout MECs in a cleared mammary fat pad of immune-deficient recipient mice resulted in development of new but dilated virgin ducts with a disrupted myoepithelial and luminal epithelial cell multilayer and aberrant ductal morphogenesis during pregnancy. In the absence of FAK, MECs spread poorly, showed enhanced Rho kinase (ROCK)-mediated cytoskeletal contractility, and failed to respond to receptor-mediated cytoskeletal remodeling. Likewise, FAK deficiency fully inhibited branching morphogenesis of mammary gland organoids in a ROCK-dependent manner. Altogether these data suggest a model in which FAK coordinates contractile forces in MECs to maintain the bilayered cellular organization of myoepithelial and luminal epithelial cells in ducts, thus allowing proper mammary gland development and function.

Disrupted expression of CXCL5 in colorectal cancer is associated with rapid tumor formation in rats and poor prognosis in patients.

PURPOSE: We isolated a subline (CC531M) from the CC531S rat colon carcinoma cell line, which grows and metastasizes much more rapidly than CC531S. We found, using RNA expression profiling, that one of the major changes in the CC531M cell line was a 5.8-fold reduction of the chemokine CXCL5. The purpose of this study was to determine the effect of CXCL5 expression on colorectal tumor growth and metastasis. EXPERIMENTAL DESIGN: CC531 clones were generated with either knockdown or restored expression of CXCL5. These clones were inoculated in the liver of rats. In addition, in two independent cohorts of colorectal cancer patients, the level of CXCL5 expression was determined and associated to clinical variables. RESULTS: Knockdown of CXCL5 expression in CC531S resulted in rapid tumor growth and increased number of metastasis, whereas restored expression of CXCL5 in CC531M resulted in a return of the "mild" tumor growth pattern of the parental cell line CC531S. In vitro, no difference was found in proliferation rate between clones with either high or low expression of CXCL5, suggesting that environmental interactions directed by CXCL5 determine tumor outgrowth. Finally, the importance of our findings was established for patients with colorectal cancer. We found that low expression of CXCL5 was significantly associated with poor prognosis for colorectal cancer patients. CXCL5 showed a trend (P = 0.05) for a positive correlation with intratumoral CD8(+) T-cell infiltration, suggesting a possible explanation for the observed poorer prognosis. CONCLUSIONS: Our results show that CXCL5 is important in growth and development of colorectal cancer, implicating a future role in both cancer therapy and diagnosis.

FRET biosensor-based kinase inhibitor screen for ERK and AKT activity reveals differential kinase dependencies for proliferation in TNBC cells.

Enhanced expression and activity of protein kinases are critical in tumor cell proliferation and cancer progression. These various cancer-related kinases form intricate interdependent signaling networks. Evaluation of the effect of various kinase inhibitors on these networks is critical to understand kinase inhibitor efficacy in cancer therapy. The dynamic activation of some kinases can be monitored by fluorescence resonance energy transfer (FRET) biosensors with high temporal resolution. Here, we established a FRET biosensor-based high throughput imaging approach to determine ERK and AKT activity in two triple negative breast cancer (TNBC) cell lines HCC1806 and Hs578T. FRET functionality was systematically evaluated using EGF stimulation and different MEK and AKT inhibitors, respectively. Next, we assessed the effect of a kinase inhibitor library containing >350 different kinase inhibitors (KIs) on ERK and AKT kinase activity using a FRET high-throughput screening setting. Suppression of FRET-ERK activity was generally positively correlated with the proliferation phenotype against inhibitors targeting MAPK signaling in both cell lines containing FRET-ERK reporter. AKT inhibitor (AKTi) resistant HCC1806 showed decreased proliferation associated with downregulated dynamics of FRET-ERK when treated with KIs targeting protein receptor tyrosine kinase (RTK). Yet, MEK inhibitor (MEKi) resistant Hs578T showed positively correlated FRET-AKT and proliferative responses against different PI3K and AKT inhibitors. Altogether, our data demonstrate the feasibility to integrate high throughput imaging-based screening of intracellular kinase activity using FRET-based biosensors in assessing kinase specificity and possible signaling crosstalk in direct relation to therapeutic outcome.

Uncovering the signaling landscape controlling breast cancer cell migration identifies novel metastasis driver genes.

Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug development.

Caspase-3 activity predicts local recurrence in rectal cancer.

PURPOSE: Radiotherapy followed by total mesorectal excision surgery has been shown to significantly reduce local recurrence rates in rectal cancer patients. Radiotherapy, however, is associated with considerable morbidity. The present study evaluated the use of biochemical detection of enzymatic caspase-3 activity as preoperative marker for apoptosis to preselect patients that are unlikely to develop a local recurrence to spare these patients from overtreatment and the negative side effects of radiotherapy. EXPERIMENTAL DESIGN: Nonirradiated freshly frozen tissue samples from 117 stage III rectal cancer patients were collected from a randomized clinical trial that evaluated preoperative radiotherapy in total mesorectal excision surgery. Additional frozen archival tissues from 47 preoperative biopsies and corresponding resected colorectal tumors were collected. Level of apoptosis was determined by measuring the enzymatic activity of caspase-3 in a biochemical assay. RESULTS: In tumor tissue, caspase-3 activity lower than the median was predictive of 5-year local recurrence (hazard ratio, 7.4; 95% confidence interval, 1.7-32.8; P = 0.008), which was unaffected by adjustment for type of resection, tumor location, and T status (adjusted hazard ratio, 7.5; 95% confidence interval, 1.7-34.1; P = 0.009). Caspase-3 activity in preoperative biopsies was significantly correlated with caspase-3 activity in corresponding resected tumors (r = 0.56; P < 0.0001). CONCLUSION: Detection of tumor apoptosis levels by measuring caspase-3 activity, for which a preoperative biopsy can be used, accurately predicted local recurrence in rectal cancer patients. These findings indicate that caspase-3 activity is an important denominator of local recurrence and should be evaluated prospectively to be added to the criteria to select rectal cancer patients in which radiotherapy is redundant.

Impaired activation of caspases and prevention of mitochondrial dysfunction in the metastatic colon carcinoma CC531s-m2 cell line.

In a previous paper we described the properties of a rapidly metastasizing cell line CC531s-m2 derived from the poorly metastasizing CC531s cell. The m2-cell line was relatively resistant to killing by NK cells. Both CD95L and TRAIL mediated apoptosis was decreased in the m2-cell line. Now, by flow cytometrical analysis of intra- and extra-cellular expressed receptors, we show that the localization of the receptors for CD95L and TRAIL was not altered in the CC531s-m2 cells as compared to the parental cell line. Subsequently caspase-activation and mitochondrial function were studied by enzymatic cleavage of fluorescent caspase-substrates and retention of the mitochondrial dye rhodamine-123, respectively. The activation of caspases as well as the loss of the mitochondrial membrane potential (MMP) was less in the CC531s-m2 cell line upon CD95L- and TRAIL-signalling. Furthermore, the sensitivity of the CC531-m2 towards cisplatin-induced apoptosis was strongly decreased. This was consistent with less mitochondrial damage, delayed caspase cleavage and decreased caspase activity. Altogether, we conclude that an Natural Killer-cell insensitive cell is less sensitive to CD95L- and TRAIL-induced apoptosis as well as anti-cancer drug induced apoptosis by prevention of mitochondrial damage and activation of caspases.

SYK is a candidate kinase target for the treatment of advanced prostate cancer.

Improved targeted therapies are needed to combat metastatic prostate cancer. Here, we report the identification of the spleen kinase SYK as a mediator of metastatic dissemination in zebrafish and mouse xenograft models of human prostate cancer. Although SYK has not been implicated previously in this disease, we found that its expression is upregulated in human prostate cancers and associated with malignant progression. RNAi-mediated silencing prevented invasive outgrowth in vitro and bone colonization in vivo, effects that were reversed by wild-type but not kinase-dead SYK expression. In the absence of SYK expression, cell surface levels of the progression-associated adhesion receptors integrin α2ß1 and CD44 were diminished. RNAi-mediated silencing of α2ß1 phenocopied SYK depletion in vitro and in vivo, suggesting an effector role for α2ß1 in this setting. Notably, pharmacologic inhibitors of SYK kinase currently in phase I-II trials for other indications interfered similarly with the invasive growth and dissemination of prostate cancer cells. Our findings offer a mechanistic rationale to reposition SYK kinase inhibitors for evaluation in patients with metastatic prostate cancer.

Rat colon carcinoma cells that survived systemic immune surveillance are less sensitive to NK-cell mediated apoptosis.

In order to form distant metastases, cells from the primary tumor have to detach, enter the blood- or lymph-compartment and escape immune surveillance. Here, we describe the selection of rat colon carcinoma cell lines (CC531s-m1 and CC531s-m2) that escaped from systemic immune surveillance; CC531s cells were injected into the v. jugularis of Wag/Rij rats, after three weeks the lung tumors were isolated, the tumor cells were cultured, characterized and injected again. The m1- and m2-cell lines were less susceptible for killing by syngeneic NK cells. Further characterization of this cell line showed a decreased sensitivity towards TRAIL- and CD95L-, but not to granzyme B-mediated apoptosis. In the m1- and m2-cells log-phase growth started earlier as compared to the parental cell line, whereas no changes were found in anchorage-dependent or anchorage-independent growth. After subcapsular injection of the m2-cell line into the liver of rats much more lung metastases were formed in comparison to injection of the parental cell line. In conclusion, the results suggest that the resistance of the m1- and m2-cells to NK cell-mediated apoptosis was associated with their capability to survive systemic immune surveillance and form metastases in vivo.

Interleukin-2 activated NK cells do not use the CD95L- and TRAIL-pathways in the rapid induction of apoptosis of rat colon carcinoma CC531s cells.

Natural Killer (NK) cells can induce apoptosis in target cells in at least four ways: by secretion of granzyme B/perforin (GrB/P) and via the CD95L, TRAIL and TNF-alpha pathways. In this study we examined the pathways used by interleukin-2 activated rat NK (A-NK) cells to induce apoptosis in the rat colon carcinoma cell line CC531s. Co-incubation of A-NK cells with CC531s cells for three hours resulted in 70% apoptosis in the latter. Addition of the GrB/P pathway-inhibitor concanamycin A reduced the number of apoptotic cells to 54%. Blockade of the CD95L, TRAIL and TNF-alpha pathways by specific antibodies hardly had an additional effect. However, co-incubation with transfected MEC cells that expressed CD95L or 2PK3-cells that expressed TRAIL did induce apoptosis in CC531s cells. Furthermore the A-NK cells contained CD95L and TRAIL. However, comparison of non- and permeabilized cells revealed that the majority of TRAIL was present in the cytosol of A-NK cells and was not available for induction of apoptosis. The presence of elevated levels of bcl-2 in CC531 cells reduced the sensitivity towards induction of apoptosis both by A-NK cells as well as the CD95L and TRAIL expressing cell lines. Using the caspase-inhibitors ac-IEPD-CHO, ac-DEVD-CHO and zVAD-fmk, it was shown that inhibition of the effector caspase-3 prevented A-NK cell induced apoptosis in CC531-bcl-2 cells, but not in CC531s cells. In conclusion, A-NK cells kill by secretion of GrB/P and not by the CD95L, TRAIL or TNF pathways albeit both CD95L and TRAIL are produced by the A-NK cells.

The role of calpains in apoptotic changes in isolated hepatocytes after attack by Natural Killer cells.

Previously, we showed that interleukin-2 activated Natural Killer cells (A-NK cells) in vitro rapidly induced apoptosis in freshly isolated rat hepatocytes (Blom et al., 1999. Hepatology 29 (3): 785-792) which was caused by a rapid decrease in the mitochondrial membrane potential and activation of caspases. In the present study we investigated the involvement of calpains in A-NK cell-induced apoptosis in isolated hepatocytes. When NK cells and hepatocytes were incubated in the presence of a calpain inhibitor the number of apoptotic cells decreased from 46 to 36%. However, more hepatocytes became necrotic (48 vs. 30%) as compared to the uninhibited situation. Inhibition of the calpains alone could not prevent the induction of the nuclear and cytoskeletal disruptions occurring in the hepatocytes. Inhibition of both calpains and caspases increased the number of necrotic cells as compared to incubation with a single inhibitor. However, the damage to the cytoskeleton of the surviving cells was completely inhibited. We conclude that calpains play a role in induction of apoptosis by NK cells. However, their role is limited as compared to caspases.

Drug-induced endoplasmic reticulum and oxidative stress responses independently sensitize toward TNFα-mediated hepatotoxicity.

Drug-induced liver injury (DILI) is an important clinical problem. Here, we used a genomics approach to in detail investigate the hypothesis that critical drug-induced toxicity pathways act in synergy with the pro-inflammatory cytokine tumor necrosis factor α (TNFα) to cause cell death of liver HepG2 cells. Transcriptomics of the cell injury stress response pathways initiated by two hepatoxicants, diclofenac and carbamazepine, revealed the endoplasmic reticulum (ER) stress/translational initiation signaling and nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) antioxidant signaling as two major affected pathways, which was similar to that observed for the majority of ∼80 DILI compounds in primary human hepatocytes. Compounds displaying weak or no TNFα synergism, namely ketoconazole, nefazodone, and methotrexate, failed to synchronously induce both pathways. The ER stress induced was primarily related to protein kinase R-like ER kinase (PERK) and activating transcription factor 4 (ATF4) activation and subsequent expression of C/EBP homologous protein (CHOP), which was all independent of TNFα signaling. Identical ATF4 dependent transcriptional programs were observed in primary human hepatocytes as well as primary precision-cut human liver slices. Targeted RNA interference studies revealed that whereas ER stress signaling through inositol-requiring enzyme 1α (IRE1α) and activating transcription factor 6 (ATF6) acted cytoprotective, activation of the ER stress protein kinase PERK and subsequent expression of CHOP was pivotal for the onset of drug/TNFα-induced apoptosis. Whereas inhibition of the Nrf2-dependent adaptive oxidative stress response enhanced the drug/TNFα cytotoxicity, Nrf2 signaling did not affect CHOP expression. Both hepatotoxic drugs enhanced expression of the translational initiation factor EIF4A1, which was essential for CHOP expression and drug/TNFα-mediated cell killing. Our data support a model in which enhanced drug-induced translation initiates PERK-mediated CHOP signaling in an EIF4A1 dependent manner, thereby sensitizing toward caspase-8-dependent TNFα-induced apoptosis.

Automated whole animal bio-imaging assay for human cancer dissemination.

A quantitative bio-imaging platform is developed for analysis of human cancer dissemination in a short-term vertebrate xenotransplantation assay. Six days after implantation of cancer cells in zebrafish embryos, automated imaging in 96 well plates coupled to image analysis algorithms quantifies spreading throughout the host. Findings in this model correlate with behavior in long-term rodent xenograft models for panels of poorly- versus highly malignant cell lines derived from breast, colorectal, and prostate cancer. In addition, cancer cells with scattered mesenchymal characteristics show higher dissemination capacity than cell types with epithelial appearance. Moreover, RNA interference establishes the metastasis-suppressor role for E-cadherin in this model. This automated quantitative whole animal bio-imaging assay can serve as a first-line in vivo screening step in the anti-cancer drug target discovery pipeline.

Two-photon intravital multicolor imaging combined with inducible gene expression to distinguish metastatic behavior of breast cancer cells in vivo.

PURPOSE: The aim of this study is to use multicolor intravital imaging together with an inducible cell model to compare metastatic behavior of control and genetically modified breast cancer cell populations within the intact primary tumor of a mouse. PROCEDURE: GFP-MTLn3-ErbB1 cells were generated with doxycycline-regulated conditional transgene expression using lentiviral TREAutoR3-cyan fluorescent protein (CFP). CFP expression together with tumor cell motility is monitored in vitro and in vivo. RESULTS: Effective and tight control of doxycycline-induced CFP expression was observed both in vitro and in vivo. Intravital multiphoton microscopy on intact orthotopic tumors allowed a clear discrimination between GFP-only and (GFP + CFP) cell populations, which enables direct comparison of the motility behavior of two different cell populations in the same microenvironment in vivo. CONCLUSIONS: This system is robust and versatile for conditional gene expression and can be used to study the role of individual candidate metastasis genes in vitro and in vivo. This technology will allow investigations of cellular events in cancer metastasis and in particular intravasation within a primary tumor.

Two-photon intravital multicolour imaging to study metastatic behaviour of cancer cells in vivo.

In the last decade, intravital microscopy on breast tumours in mice at single-cell resolution has resulted in important new insight into mechanisms of metastatic behaviour such as migration, invasion, and intravasation of tumour cells; angiogenesis; and the response of immune cells. This chapter describes the methods that can be used for analysing tumour cell motility in a mouse model of breast cancer metastasis. It includes protocols for generation of a labelled primary tumour, its imaging with two-photon microscopy, and the processing of time-lapse image data. Furthermore, we present a methodology, recently developed in our laboratory that combines multicolour imaging with an inducible cell model to study the role of a specific gene of interest in tumour cell motility in vivo. This protocol can be used to image the metastatic behaviour of different individual tumour cells within the same tumour microenvironment and correlate it with metastasis formation. Additional protocols for labelling macrophages to visualise blood flow and image analysis are also included.