Transcriptional Induction of SERPINE1 and Fibrinolysis Inhibition as Predominant Effects of Glucocorticoids on the Cancer Coagulome.

BACKGROUND/AIM: How tumors regulate the genes of the coagulome is crucial for cancer-associated thrombosis and the occurrence of venous thromboembolic complications in patients with cancer. We have previously reported potent yet complex effects of glucocorticoids (GC) on the expression of three genes that play a key role in the regulation of thrombin/plasmin activation (F3, PLAU, and SERPINE1). This study aimed to extend the investigation of GC effects to the whole tumor coagulome and assess the resulting impact on the ability of cancer cells to activate thrombin and plasmin. MATERIALS AND METHODS: Cancer RNA expression data were retrieved from various sources. Additionally, oral squamous cell carcinoma (OSCC) cells exposed to GC in vitro were analyzed using QPCR, enzymatic assays measuring thrombin and urokinase-type Plasminogen Activator (uPA) activity, and D-dimer concentrations. RESULTS: Our findings highlight the potent and specific stimulatory effect of GC on SERPINE1 expression across different types of cancer. Consistently, GC were found to inhibit uPA proteolytic activity and reduce the concentrations of D-dimers in OSCC in vitro. CONCLUSION: Fibrinolysis inhibition is a key consequence of cancer cell exposure to GC, possibly leading to the stabilization of the fibrin clot in cancer.

The Tumor Coagulome as a Transcriptional Target and a Potential Effector of Glucocorticoids in Human Cancers.

BACKGROUND: The coagulome, defined as the repertoire of genes that locally regulate coagulation and fibrinolysis, is a key determinant of vascular thromboembolic complications of cancer. In addition to vascular complications, the coagulome may also regulate the tumor microenvironment (TME). Glucocorticoids are key hormones that mediate cellular responses to various stresses and exert anti-inflammatory effects. We addressed the effects of glucocorticoids on the coagulome of human tumors by investigating interactions with Oral Squamous Cell Carcinoma, Lung Adenocarcinoma, and Pancreatic Adenocarcinoma tumor types. METHODS: We analyzed the regulation of three essential coagulome components, i.e., the tissue factor (TF), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1) in cancer cell lines exposed to specific agonists of the glucocorticoid receptor (GR) (dexamethasone and hydrocortisone). We used QPCR, immunoblots, small-interfering RNA, Chromatin immunoprecipitation sequencing (ChIPseq) and genomic data from whole tumor and single-cell analyses. RESULTS: Glucocorticoids modulate the coagulome of cancer cells through a combination of indirect and direct transcriptional effects. Dexamethasone directly increased PAI-1 expression in a GR-dependent manner. We confirmed the relevance of these findings in human tumors, where high GR activity/high SERPINE1 expression corresponded to a TME enriched in active fibroblasts and with a high TGF-ß response. CONCLUSION: The transcriptional regulation of the coagulome by glucocorticoids that we report may have vascular consequences and account for some of the effects of glucocorticoids on the TME.

Analysis of Mesencephalic Astrocyte-derived Neurotrophic Factor in Multiple Myeloma.

BACKGROUND/AIM: Multiple myeloma (MM) is characterized by high production of immunoglobulins resulting in a constant source of endoplasmic reticulum (ER)-stress. Mesencephalic astrocyte-derived neurotrophic factor (MANF) was identified as a possible circulating biomarker that could help in monitoring ER-stress mediated diseases. MATERIALS AND METHODS: To assess the relevance of MANF in MM, we performed in silico and in vitro analysis in malignant cell lines including the myeloma cell line RPMI 8226. Serum MANF concentration was compared between healthy subjects (n=60), patients with MM (n=68), or those with monoclonal gammopathy of undetermined significance (MGUS) (n=73). RESULTS: MANF mRNA expression was upregulated in the RPMI 8226 cell line, and higher secretion of MANF was measured in RPMI 8226 supernatant. Serum MANF levels were not significantly different between MM or MGUS patients and those in age- and sex-matched healthy controls. CONCLUSION: MANF was not validated as a biomarker of interest in MM patients. Its potential implication in myeloma pathogenesis should be investigated.

DNA damage response- and JAK-dependent regulation of PD-L1 expression in head and neck squamous cell carcinoma (HNSCC) cells exposed to 5-fluorouracil (5-FU).

OBJECTIVES: The immune checkpoint molecule PD-L1 (CD274) is a crucial regulator of the tumor immune response. Its expression has been reported in the therapeutic context in Head and Neck Squamous Cell Carcinoma (HNSCC), but it remains unclear how therapeutically approved molecules regulate PD-L1 expression in HNSCC cells. MATERIALS AND METHODS: Three HNSCC cell lines (BICR6, PE/CA-PJ34 and PE/CA-PJ41) were used to analyze PD-L1 expression by immunoblotting, immunofluorescence and QPCR. Freely-available single cell RNAseq data from HNSCC were also used. RESULTS: 5-Fluorouracil (5-FU) increased the expression of PD-L1 with high efficacy in HNSCC cells. Single cell RNAseq data suggested the specificity of the regulation of PD-L1 in this context. The effect of 5-FU on PD-L1 expression was related to its genotoxic effect and was prevented by extracellular application of thymidine or using a chemical inhibitor of the DNA damage Response kinases ATM/ATR. We found that the effect of 5-FU was additive or synergistic with IFN-γ, the canonical inducer of PD-L1 in epithelial cells. QPCR analysis confirmed this finding and identified JAK-dependent transcriptional activation of PD-L1/CD274 as the underlying mechanism. The induction of PD-L1 by 5-FU was partially prevented by Epidermal Growth Factor Receptor (EGFR) inhibition with cetuximab. CONCLUSION: Our study highlights the specific DNA Damage Response- and JAK- dependent induction of PD-L1 by 5-FU in HNSCC cells. This induction is regulated by the cytokine context and is potentially therapeutically actionable.

SLC7A11 as a biomarker and therapeutic target in HPV-positive head and neck Squamous Cell Carcinoma.

Ferroptosis, a regulated form of cell necrosis was previously reported to be induced upon pharmacological targeting of the cystine transporter SLC7A11 in Head and neck Squamous Cell Carcinoma (HNSCC). Whether tumors arising in a context of chronic infection with Human Papillomavirus (HPV) are sensitive to ferroptosis is unknown. Using RNAseq data (both whole-tumor and single-cell sequencing) we report that HPV positive (HPV+ve) tumors have lower expression levels of SLC7A11 compared to HPV negative (HPV-ve) HNSCC. We examined in vitro the effect of erastin, a specific blocker of SLC7A11, applied on two HNSCC cell lines with stable expression of HPV16 E6 and E7 oncoproteins. We report a decrease in total GSH levels and an increased sensitivity to erastin-induced ferroptosis in E6-E7 cells. Cell sensitivity to ferroptosis was specificaly related to a defect in cystine transport since we found no difference in ferroptosis induced by the direct inhibition of GPX4, and N-Acetyl Cystein abolished the difference between WT and E6-E7-expressing cells. Our findings point to SLC7A11 as an HPV-related biomarker of potential therapeutic relevance in HNSCC. Targeting cystine import to promote ferroptosis might be a promising strategy against HPV+ve HNSCC. (188 words).

ERK1/2 signaling regulates the immune microenvironment and macrophage recruitment in glioblastoma.

The tumor microenvironment is an important determinant of glioblastoma (GBM) progression and response to treatment. How oncogenic signaling in GBM cells modulates the composition of the tumor microenvironment and its activation is unclear. We aimed to explore the potential local immunoregulatory function of ERK1/2 signaling in GBM. Using proteomic and transcriptomic data (RNA seq) available for GBM tumors from The Cancer Genome Atlas (TCGA), we show that GBM with high levels of phosphorylated ERK1/2 have increased infiltration of tumor-associated macrophages (TAM) with a non-inflammatory M2 polarization. Using three human GBM cell lines in culture, we confirmed the existence of ERK1/2-dependent regulation of the production of the macrophage chemoattractant CCL2/MCP1. In contrast with this positive regulation of TAM recruitment, we found no evidence of a direct effect of ERK1/2 signaling on two other important aspects of TAM regulation by GBM cells: (1) the expression of the immune checkpoint ligands PD-L1 and PD-L2, expressed at high mRNA levels in GBM compared with other solid tumors; (2) the production of the tumor metabolite lactate recently reported to dampen tumor immunity by interacting with the receptor GPR65 present on the surface of TAM. Taken together, our observations suggest that ERK1/2 signaling regulates the recruitment of TAM in the GBM microenvironment. These findings highlight some potentially important particularities of the immune microenvironment in GBM and could provide an explanation for the recent observation that GBM with activated ERK1/2 signaling may respond better to anti-PD1 therapeutics.

Early decrease in serum amphiregulin or vascular endothelial growth factor levels predicts sorafenib efficacy in hepatocellular carcinoma.

Sorafenib is the standard of care for the treatment of advanced hepatocellular carcinoma (HCC). However, identifying secreted biomarkers that predict sorafenib efficacy in all HCC patients remains challenging. It was recently reported that sorafenib interferes with protein homeostasis and inhibits global translation in tumour cells. A likely consequence of this inhibition would be the interruption of autocrine loops. The aim of the present study was to investigate the effect of sorafenib on two growth factors implicated in autocrine loops and HCC tumour invasion: amphiregulin (AREG) and vascular endothelial growth factor (VEGF). ELISA, quantitative polymerase chain reaction analysis, western blotting and a cytokine array were performed on HCC cell lines and the prognostic role of these two biomarkers in HCC patients was evaluated. Serum AREG and VEGF levels were assayed by ELISA in 55 patients with advanced HCC treated with sorafenib. It was observed that sorafenib decreased AREG, VEGF and cytokine expression at the transcriptional and post­transcriptional levels. All HCC patients in our cohort had detectable concentrations of AREG and VEGF both at baseline and after sorafenib treatment. The decreased serum levels of AREG and VEGF after 15 days of sorafenib treatment were significantly associated with better overall and progression­free survival. The results of the multivariate analysis demonstrated that a decrease in AREG was an independent prognostic indicator of overall survival (hazard ratio, 0.208; 95% confidence interval, 0.173­0.673; P=0.0003). These results suggest that sorafenib inhibits auto-crine loops and that early decrease in serum AREG or VEGF levels predicts sorafenib efficacy in HCC patients.

A pan-cancer study of the transcriptional regulation of uricogenesis in human tumours: pathological and pharmacological correlates.

Uric acid (UA) is the end product of the catabolism of purines, and its serum levels are commonly increased in cancer patients. We aimed to explore the transcriptional regulation of tumour uricogenesis in human tumours, and relate uricogenesis with tumour pathological and pharmacological findings. Using data from The Cancer Genome Atlas (TCGA), we analysed the expression levels of xanthine dehydrogenase (XDH) and adenine phosphoribosyltransferase (APRT), two key enzymes in UA production and the purine salvage pathway, respectively. We found large differences between tumour types and individual tumours in their expression of XDH and APRT Variations in locus-specific DNA methylation and gene copy number correlated with the expression levels of XDH and APRT in human tumours respectively. We explored the consequences of this differential regulation of uricogenesis. Tumours with high levels of XDH mRNA were characterised by higher expression of several genes encoding pro-inflammatory and immune cytokines, and increased levels of tumour infiltration with immune cells. Finally, we studied cancer drug sensitivity using data from the National Cancer Institute-60 (NCI-60) database. A specific correlation was found between the expression levels of APRT and cell sensitivity to the chemotherapeutic agent 5-fluorouracil (5-FU). Our findings underline the existence of great differences in uricogenesis between different types of human tumours. The study of uricogenesis offers promising perspectives for the identification of clinically relevant molecular biomarkers and for tumour stratification in the therapeutic context.

Protein biosynthesis, a target of sorafenib, interferes with the unfolded protein response (UPR) and ferroptosis in hepatocellular carcinoma cells.

Sorafenib is the first line treatment for advanced hepatocellular carcinoma (HCC). We explored its impact on the proteostasis of cancer cells, i.e. the processes that regulate the synthesis, maturation and turn-over of cellular proteins. We observed that sorafenib inhibits the production of the tumour marker alpha-foetoprotein (AFP) in two different HCC cell lines, an effect that correlated with a radical inhibition of protein biosynthesis. This effect was observed at clinically relevant concentrations of sorafenib and was not related to the effect of sorafenib on the transport of amino acids across the plasma membrane or the induction of the unfolded protein response (UPR). Instead, we observed that sorafenib inhibits translation initiation and the mechanistic target of rapamycin (mTOR) signaling cascade, as shown by the analysis of phosphorylation levels of the protein 4EBP1 (eukaryotic translation initiation factor 4E binding protein 1). We explored the consequences of this inhibition in HCC cells. We observed that overall sorafenib is a weak inducer of the UPR that can paradoxically prevent the UPR induced by tunicamycin. We also found no direct synergistic anticancer effect between sorafenib and various strategies that inhibit the UPR. In agreement with the possibility that translation inhibition might be an adaptive stress response in HCC cells, we noted that it protects cancer cell from ferroptosis, a form of oxidative necrosis. Our findings point to the modulation of protein biosynthesis and mTOR signaling as being important, yet complex determinants of the response of HCC cells to sorafenib.

Targeting the Unfolded Protein Response as a Potential Therapeutic Strategy in Renal Carcinoma Cells Exposed to Cyclosporine A.

BACKGROUND/AIM: Organ transplant patients treated with the immunosuppressive drug cyclosporine A often present malignant kidney tumors. Cyclosporine A can promote oncogenesis in a cell-intrinsic manner by increasing the production of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: We explored the impact of cyclosporine A and the role of the unfolded protein response (UPR) on three human renal cell carcinoma (RCC) cell lines under normoxic and hypoxic (1% O2) conditions. RESULTS: Cyclosporine A regulated the expression of VEGF at the post-transcriptional level. Cyclosporine A induced the inositol requiring enzyme-1α (IRE1α) arm of the UPR and stabilized neosynthesized proteins in RCC cells. Toyocamycin, an inhibitor of IRE1α, abolished the clonogenic growth of RCC cells and reduced induction of VEGF by cyclosporine A under hypoxia. CONCLUSION: Our findings highlight the impact of cyclosporine A on the proteostasis of RCC cells, and suggest the potential therapeutic interest of targeting the UPR against tumors arising in the context of organ transplantation.

Mathematical modelling unveils the essential role of cellular phosphatases in the inhibition of RAF-MEK-ERK signalling by sorafenib in hepatocellular carcinoma cells.

The RAS-RAF-MEK-ERK cascade is a key oncogenic signal transduction pathway activated in many types of tumours in humans. Sorafenib, the medical treatment of reference against advanced stages of hepatocellular carcinoma (HCC), inhibits the RAF-MEK-ERK cascade in HCC cells. Based on previous studies suggesting that this cascade is an important target of sorafenib in HCC cells, we explored its regulation using mathematical modelling and ordinary differential equations. We analysed the dynamic regulation of the core components of the RAF-MEK-ERK cascade in three human HCC cell lines (Huh7, Hep3B and PLC/PRF5) with heterogeneous responses to sorafenib. In silico predictions derived from our mathematical model suggested that the disappearance of phosphorylated MEK and ERK proteins catalysed by cellular phosphatases is an essential mechanism underlying the anti-ERK efficacy of sorafenib in HCC cells. This prediction was experimentally validated using specific inhibitors of the phosphatases PP2A (Protein Phosphatase 2A) and DUSP1/6 (Dual-specificity phosphatases 1/6). These findings highlight an unexpected mode of action of sorafenib on the kinome of HCC cells, and open new perspectives regarding the therapeutic targeting of the RAF-MEK-ERK cascade in this context.

Metallothionein-1 as a biomarker of altered redox metabolism in hepatocellular carcinoma cells exposed to sorafenib.

BACKGROUND: Sorafenib, a kinase inhibitor active against various solid tumours, induces oxidative stress and ferroptosis, a new form of oxidative necrosis, in some cancer cells. Clinically-applicable biomarkers that reflect the impact of sorafenib on the redox metabolism of cancer cells are lacking. METHODS: We used gene expression microarrays, real-time PCR, immunoblot, protein-specific ELISA, and gene reporter constructs encoding the enzyme luciferase to study the response of a panel of cancer cells to sorafenib. Tumour explants prepared from surgical hepatocellular carcinoma (HCC) samples and serum samples obtained from HCC patients receiving sorafenib were also used. RESULTS: We observed that genes of the metallothionein-1 (MT1) family are induced in the HCC cell line Huh7 exposed to sorafenib. Sorafenib increased the expression of MT1G mRNA in a panel of human cancer cells, an effect that was not observed with eight other clinically-approved kinase inhibitors. We identified the minimal region of the MT1G promoter that confers inducibility by sorafenib to a 133 base pair region containing an Anti-oxidant Response Element (ARE) and showed the essential role of the transcription factor NRF2 (Nuclear factor erythroid 2-Related Factor 2). We examined the clinical relevance of our findings by analysing the regulation of MT1G in five tumour explants prepared from surgical HCC samples. Finally, we showed that the protein levels of MT1 increase in the serum of some HCC patients receiving sorafenib, and found an association with reduced overall survival. CONCLUSION: These findings indicate that MT1 constitute a biomarker adapted for exploring the impact of sorafenib on the redox metabolism of cancer cells.

Personalization of the medical treatment of solid tumours using patient-derived tumour explants (Review).

Improving the pre-clinical characterization of therapeutic approaches and developing new biological assays that will enable treatment personalization for individual patients are promising developments in oncology. Here we describe a new approach consisting of culturing human tumour explants. This approach involves the preparation of slices from freshly-obtained, surgically-resected material that can be maintained ex vivo for several days. Recent studies have provided proof of principle that this approach can be easily implemented in order to explore the mode of action of various anticancer drugs and the responses of 'real' tumours at the individual patient level. We present the practical aspects and highlight the versatility of this approach, which allows for the analysis of the susceptibility of any individual tumour to multiple anticancer drugs in parallel. We discuss its potential as a companion assay in the design of optimal clinical trials and as a guide for the prescription of medical treatment. We discuss which future clinical and biological studies are needed to validate the information gathered from cultured tumour explants, and to integrate this information with that gathered from other assays in order to optimize the medical treatment of cancer.

Alpha-fetoprotein is a biomarker of unfolded protein response and altered proteostasis in hepatocellular carcinoma cells exposed to sorafenib.

Sorafenib is the treatment of reference for advanced hepatocellular carcinoma (HCC). A decrease in the serum levels of Alpha-fetoprotein (AFP) is reported to be the biological parameter that is best associated with disease control by sorafenib. In order to provide a biological rationale for the variations of AFP, we analyzed the various steps of AFP production in human HCC cell lines exposed to sorafenib. Sorafenib dramatically reduced the levels of AFP produced by HCC cells independently of its effect on cell viability. The mRNA levels of AFP decreased upon sorafenib treatment, while the AFP protein remained localized in the Golgi apparatus. Sorafenib activated the Regulated Inositol-Requiring Enzyme-1α (IRE-1α) and the PKR-like ER Kinase (PERK)-dependent arms of the Unfolded Protein Response (UPR). The inhibition of IRE-1α partially restored the mRNA levels of AFP upon treatment with sorafenib. The inhibition of both pathways partially prevented the drop in the production of AFP induced by sorafenib. The findings provide new insights on the regulation of AFP, and identify it as a biomarker suitable for the exploration of HCC cell proteostasis in the context of therapeutic targeting.

The kinase-inhibitor sorafenib inhibits multiple steps of the Hepatitis C Virus infectious cycle in vitro.

Hepatitis C Virus (HCV) chronic infection is a major cause of hepatocellular carcinoma. Sorafenib is the only medical treatment that has been approved for the treatment of this cancer. It is a multikinase inhibitor with anti-tumor activity against a wide variety of cancers. Sorafenib blocks angiogenesis and tumor cell proliferation through inhibition of kinases, such as VEGFR2, PDGFR, or the serine/threonine kinases RAF. Previous studies have reported an anti-HCV effect of sorafenib in vitro, but various mechanisms of action have been described. The aim of this study was to clarify the action of sorafenib on the complete HCV infectious cycle. In order to examine the action of sorafenib on all steps of the HCV infectious cycle, we used a combination of validated cell culture models, based on the HuH-7 reference cell line and primary human hepatocytes. We found that sorafenib blocks HCV infection by altering the viral entry step and the production of viral particles. Moreover, we observed that treatment with sorafenib lead to a modification of Claudin-1 expression and localization, which could partly be responsible for the anti-HCV effect. Collectively, our findings confirm the anti-HCV effect of sorafenib in vitro, while highlighting the complexity of the action of sorafenib on the HCV infectious cycle.

Biomarkers of apoptosis and necrosis in patients with hepatocellular carcinoma treated with sorafenib.

BACKGROUND/AIM: Sorafenib is the medical reference for treatment of hepatocellular carcinoma (HCC). Multiple forms of cytotoxicity are induced by sorafenib in HCC cells in vitro but it is unclear what extent of apoptosis and necrosis is induced in HCC patients receiving sorafenib. PATIENTS AND METHODS: The M30 and M65 biomarkers, which reflect the release of cytokeratin-18 and its apoptotic cleavage fragments, were measured in patients with HCC (n=36) and matched patients with cirrhosis (n=47). A serum sample was collected from 20 patients with HCC four weeks after the onset of treatment with sorafenib. RESULTS: Basal serum levels of M30 and M65 were increased in patients with HCC compared to those with uncomplicated cirrhosis. No statistically significant increase in the level of M30 or M65 was found in the sera of patients with HCC after sorafenib. CONCLUSION: The findings indicate that sorafenib is not a potent inducer of HCC cell death in most patients.

The retinoblastoma (Rb) protein regulates ferroptosis induced by sorafenib in human hepatocellular carcinoma cells.

Sorafenib is the treatment of reference for advanced hepatocellular carcinoma (HCC), the most frequent form of primary liver tumour. The loss of function of the retinoblastoma (Rb) protein is an important event during liver carcinogenesis, but it is unclear whether the Rb status modulates the response of HCC cells to sorafenib. Here, we examined this question in HCC cells with reduced levels of Rb achieved through stable RNA interference. We show that HCC cells with reduced levels of Rb exhibit a two- to threefold increase in cell death induction upon exposure to sorafenib compared with controls. Sorafenib treatment of Balb/c nude mice that received tumour xenografts derived from HCC cells with reduced Rb levels resulted in complete tumour regression in 50% of the animals treated, compared with tumour stabilization in mice that received control cells. We show that, upon exposure to sorafenib, the Rb-negative status of HCC cells promotes the occurrence of ferroptosis, a form of oxidative necrosis. The findings highlight the role of Rb in the response of HCC cells to sorafenib and the regulation of ferroptosis.

Sorafenib induces ferroptosis in human cancer cell lines originating from different solid tumors.

BACKGROUND/AIM: Ferroptosis is a recently identified form of regulated necrosis that can be experimentally induced in cancer cells with the chemical inducer erastin. Recently, we identified sorafenib, an inhibitor of oncogenic kinases, as an inducer of ferroptosis in hepatocellular carcinoma cells. Whether sorafenib is able to exert its ferroptotic activity in cancer cells originating from other tissues is presently unclear. MATERIALS AND METHODS: We compared the levels of ferroptosis induced by sorafenib with those induced by the reference compound erastin in a panel of ten human cell lines originating from various tissues. RESULTS: Sorafenib induced ferroptosis in different cancer cell lines. We found a positive correlation between the ferroptotic potency of sorafenib and erastin. Compared to other kinase inhibitors, sorafenib is the only drug that displays ferroptotic efficacy. CONCLUSION: The findings establish sorafenib as the first clinically-approved anticancer drug that can induce ferroptosis.

[Ferroptosis, a new form of cell death relevant to the medical treatment of cancer]. / La ferroptose, une nouvelle forme de mort cellulaire applicable au traitement médical des cancers.

Ferroptosis is a form of cell death that has recently been reported during exposure to erastin, a chemical compound identified in a screen for molecules able to kill cancer cells carrying an active Ras oncogene. In cells exposed to inducers of ferroptosis, a catastrophic alteration of the cellular redox metabolism occurs, resulting in massive lipid peroxidation in the plasma membrane and loss of cell viability. We present our recent observations suggesting that sorafenib, the only medical treatment with proven efficacy against hepatocellular carcinoma, induces ferroptosis, a new anti-oncogenic mode of action of this drug. The discovery of ferroptosis sheds light on the critical adaptations of the redox metabolism in cancer cells. It might also foster the discovery of new biomarkers and innovative approaches for the treatment of cancer.

Heterogeneous sensitivity of hepatocellular carcinoma to sorafenib revealed by the short-term culture of tumor fragments.

BACKGROUND/AIM: Sorafenib is currently the only medical treatment with proven efficacy against hepatocellular carcinoma (HCC). HCC cell lines display heterogeneous sensitivity to sorafenib, but little is known about the sensitivity of clinical tumors. We aimed to examine this aspect. MATERIALS AND METHODS: Using experimental tumors generated in nude mice, we set up a technique for short-term culture of HCC fragments. We applied this technique to six human HCC samples obtained from surgical resection. RESULTS: HCC fragments in culture retain their morphology and viability for at least 48 h, permitting an in vitro analysis of the effect of sorafenib on the Extracellular signal-regulated kinase (ERK) cascade. HCC exhibit heterogeneous individual responses, ranging from potent inhibition to paradoxical activation of this oncogenic cascade. CONCLUSION: Our observations highlight the heterogeneous sensitivity of HCC to sorafenib, and point to the potential interest of short-term culture of tumor fragments for personalizing the medical treatment of HCC.