Human Colorectal Liver Metastases Research Articles

Riboflavin-LSD1 axis participates in the in vivo tumor-associated macrophage morphology in human colorectal liver metastases

Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment (TME). In colorectal liver metastasis (CLM), TAM morphology correlates with prognosis, with smaller TAMs (S-TAMs) conferring a more favorable prognosis than larger TAMs (L-TAMs). However, the metabolic profile of in vivo human TAM populations remains unknown. Multiparametric flow cytometry was used to freshly isolate S- and L-TAMs from surgically resected CLM patients (n = 14S-, 14L-TAMs). Mass spectrometry-based metabolomics analyses were implemented for the metabolic characterization of TAM populations. Gene expression analysis and protein activity were used to support the biochemical effects of the enzyme–substrate link between riboflavin and (lysine-specific demethylase 1A, LSD1) with TAM morphologies. L-TAMs were characterized by a positive correlation and a strong association between riboflavin and TAM morphologies. Riboflavin in both L-TAMs and in-vitro M2 polarized macrophages modulates LSD1 protein expression and activity. The inflammatory stimuli promoted by TNFα induced the increased expression of riboflavin transporter SLC52A3 and LSD1 in M2 macrophages. The modulation of the riboflavin-LSD1 axis represents a potential target for reprogramming TAM subtypes, paving the way for promising anti-tumor therapeutic strategies.

Spatial resolution of cellular senescence dynamics in human colorectal liver metastasis.

Hepatic metastasis is a clinical challenge for colorectal cancer (CRC). Senescent cancer cells accumulate in CRC favoring tumor dissemination. Whether this mechanism progresses also in metastasis is unexplored. Here, we integrated spatial transcriptomics, 3D-microscopy, and multicellular transcriptomics to study the role of cellular senescence in human colorectal liver metastasis (CRLM). We discovered two distinct senescent metastatic cancer cell (SMCC) subtypes, transcriptionally located at the opposite pole of epithelial (e) to mesenchymal (m) transition. SMCCs differ in chemotherapy susceptibility, biological program, and prognostic roles. Mechanistically, epithelial (e)SMCC initiation relies on nucleolar stress, whereby c-myc dependent oncogene hyperactivation induces ribosomal RPL11 accumulation and DNA damage response. In a 2D pre-clinical model, we demonstrated that RPL11 co-localized with HDM2, a p53-specific ubiquitin ligase, leading to senescence activation in (e)SMCCs. On the contrary, mesenchymal (m)SMCCs undergo TGFβ paracrine activation of NOX4-p15 effectors. SMCCs display opposing effects also in the immune regulation of neighboring cells, establishing an immunosuppressive environment or leading to an active immune workflow. Both SMCC signatures are predictive biomarkers whose unbalanced ratio determined the clinical outcome in CRLM and CRC patients. Altogether, we provide a comprehensive new understanding of the role of SMCCs in CRLM and highlight their potential as new therapeutic targets to limit CRLM progression.

High-Resolution Analysis of Mononuclear Phagocytes Reveals GPNMB as a Prognostic Marker in Human Colorectal Liver Metastasis.

Patients with colorectal liver metastasis (CLM) present with heterogenous clinical outcomes and improved classification is needed to ameliorate the therapeutic output. Macrophages (Mϕ) hold promise as prognostic classifiers and therapeutic targets. Here, stemming from a single-cell analysis of mononuclear phagocytes infiltrating human CLM, we identified two Mϕ markers associated with distinct populations with opposite clinical relevance. The invasive margin of CLM was enriched in pro-inflammatory monocyte-derived Mϕ (MoMϕ) expressing the monocytic marker SERPINB2, and a more differentiated population, tumor-associated Mϕ (TAM), expressing glycoprotein nonmetastatic melanoma protein B (GPNMB). SERPINB2+ MoMϕ had an early inflammatory profile, whereas GPNMB+ TAMs were enriched in pathways of matrix degradation, angiogenesis, and lipid metabolism and were found closer to the tumor margin, as confirmed by spatial transcriptomics on CLM specimens. In a cohort of patients, a high infiltration of SERPINB2+ cells independently associated with longer disease-free survival (DFS; P = 0.033), whereas a high density of GPNMB+ cells correlated with shorter DFS (P = 0.012) and overall survival (P = 0.002). Cell-cell interaction analysis defined opposing roles for MoMϕ and TAMs, suggesting that SERPINB2+ and GPNMB+ cells are discrete populations of Mϕ and may be exploited for further translation to an immune-based stratification tool. This study provides evidence of how multi-omics approaches can identify nonredundant, clinically relevant markers for further translation to immune-based patient stratification tools and therapeutic targets. GPNMB has been shown to set Mϕ in an immunosuppressive mode. Our high dimensional analyses provide further evidence that GPNMB is a negative prognostic indicator and a potential player in the protumor function of Mϕ populations.

Clinical Impact of Herpesvirus Entry Mediator (HVEM) in Malignant Liver Tumors

Background: Herpes virus entry mediator (HVEM), also known as tumor necrosis factor receptor (TNFR) superfamily 14, regulates a variety of physiological and pathological responses in both innate and acquired immunity. Recently, HVEM is also suggested to be a critical regulator in tumor immunity. This study aimed to clarify clinical importance of HVEM in human hepatocellular carcinoma (HCC) and colorectal liver metastasis (CRLM). Methods: This study examined 150 patients with HCC and 104 patients with CRLM who underwent curative liver resection at Nara Medical University between 2000 and 2014. Immunohistochemical staining was performed using antibodies against HVEM, CD4, CD8, and CD45RO. Results: High HVEM expression was observed in 66 of 150 patients (44.0%) with HCC, and 49 of 104 patients (47.1%) with CRLM. Expression of HVEM was not associated tumor size, number of tumors, or histologic differentiation. The high-HVEM group exhibited significantly worse overall survival (OS) than the low-HVEM group (HCC: P=0.002, CRLM: P = 0.002). Multivariate analysis showed that independent poor prognostic factors of OS for HCC were high HVEM expression in HCC and tumor size >5 cm, while in CRLM, high HVEM expression in CRLM, age of 70 years or older, and having five or more tumors were prognostic factors of OS. HVEM status was inversely correlated with tumor-infiltrating CD8+ and CD45RO+ lymphocytes in both HCC and CRLM. Conclusions: Tumour-expressing HVEM might play a critical role in human HCC and CRLM, possibly through regulating immune evasion. Therefore, targeting HVEM may be a novel promising therapeutic strategy for HCC and CRLM.

Modulation of Immune Checkpoints by Chemotherapy in Human Colorectal Liver Metastases

SummaryMetastatic colorectal cancer (CRC) is a major cause of cancer-related death, and incidence is rising in younger populations (younger than 50 years). Current chemotherapies can achieve response rates above 50%, but immunotherapies have limited value for patients with microsatellite-stable (MSS) cancers. The present study investigates the impact of chemotherapy on the tumor immune microenvironment. We treat human liver metastases slices with 5-fluorouracil (5-FU) plus either irinotecan or oxaliplatin, then perform single-cell transcriptome analyses. Results from eight cases reveal two cellular subtypes with divergent responses to chemotherapy. Susceptible tumors are characterized by a stemness signature, an activated interferon pathway, and suppression of PD-1 ligands in response to 5-FU+irinotecan. Conversely, immune checkpoint TIM-3 ligands are maintained or upregulated by chemotherapy in CRC with an enterocyte-like signature, and combining chemotherapy with TIM-3 blockade leads to synergistic tumor killing. Our analyses highlight chemomodulation of the immune microenvironment and provide a framework for combined chemo-immunotherapies.

Significance of Herpesvirus Entry Mediator Expression in Human Colorectal Liver Metastasis.

Herpesvirus entry mediator (HVEM) has been suggested to play various roles in cancer biology. The authors report that HVEM expression in tumor cells is associated with a reduction in the number of tumor-infiltrating lymphocytes and a poor prognosis after surgical resection in various human gastrointestinal cancers. This study aimed to clarify the clinical significance of HVEM expression in human colorectal liver metastasis (CRLM). This study examined the cases of 104 patients with CRLM who underwent curative liver resection at Nara Medical University between 2000 and 2014. The median follow-up period was 50.2months. Immunohistochemical staining was performed using antibodies against HVEM, CD4, CD8, and CD45RO. High HVEM expression was observed in 49 patients (47.1%) with CRLM. Expression of HVEM was not associated with age, gender, administration of preoperative chemotherapy, tumor size, number of tumors, or histologic differentiation. The high-HVEM group exhibited significantly worse overall survival (OS) than the low-HVEM group (P = 0.002). Multivariate analysis showed that high HVEM expression in CRLM, age of 70years or older, and having five or more tumors are independent poor prognostic factors for OS (hazard ratio [HR], 3.35; 95% confidence interval [CI], 1.41-7.93; P = 0.006). The number of tumor-infiltrating CD8+ and CD45RO+ T cells was significantly lower in the high-HVEM group than in the low-HVEM group. High HVEM expression in primary colorectal cancer was significantly associated with synchronous CRLM, but not with metachronous CRLM. Tumor HVEM expression might play a critical role in CRLM.

Disruption of clock gene expression in human colorectal liver metastases.

The circadian timing system controls about 40 % of the transcriptome and is important in the regulation of a wide variety of biological processes including metabolic and proliferative functions. Disruption of the circadian clock could have significant effect on human health and has an important role in the development of cancer. Here, we compared the expression levels of core clock genes in primary colorectal cancer (CRC), colorectal liver metastases (CRLM), and liver tissue within the same patient. Surgical specimens of 15 untreated patients with primary CRC and metachronous CRLM were studied. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression of 10 clock genes: CLOCK, BMAL1, PER1, PER2, PER3, CRY1, CRY2, CSNK1E, TIM, TIPIN, and 2 clock-controlled genes: Cyclin-D1, and WEE1. Expression levels of 7 core clock genes were downregulated in CRLM: CLOCK (p = 0.006), BMAL1 (p = 0.003), PER1 (p = 0.003), PER2 (p = 0.002), PER3 (p < 0.001), CRY1 (p = 0.002), and CRY2 (p < 0.001). In CRC, 5 genes were downregulated: BMAL1 (p = 0.02), PER1 (p = 0.004), PER2 (p = 0.008), PER3 (p < 0.001), and CRY2 (p < 0.001). CSNK1E was upregulated in CRC (p = 0.02). Cyclin-D1 and WEE1 were both downregulated in CRLM and CRC. Related to clinicopathological factors, a significant correlation was found between low expression of CRY1 and female gender, and low PER3 expression and the number of CRLM. Our data demonstrate that the core clock is disrupted in CRLM and CRC tissue from the same patient. This disruption may be linked to altered cell-cycle dynamics and carcinogenesis.

Determination of the electrical conductivity of human liver metastases: impact on therapy planning in the radiofrequency ablation of liver tumors.

Background Radiofrequency ablation is used to induce thermal necrosis in the treatment of liver metastases. The specific electrical conductivity of a liver metastasis has a distinct influence on the heat formation and resulting tumor ablation within the tissue. Purpose To examine the electrical conductivity σ of human colorectal liver metastases and of tumor-free liver tissue in surgical specimens. Material and Methods Surgical specimens from patients with resectable colorectal liver metastases were used for measurements (size of metastases <30 mm). A four-needle measuring probe was used to determine the electrical conductivity σ of human colorectal liver metastasis (n = 8) and tumor-free liver tissue (n = 5) in a total of five patients. All measurements were performed at 470 kHz, which is the relevant frequency for radiofrequency ablation. The tissue temperature was also measured. Hepatic resections were performed in accordance with common surgical standards. Measurements were performed in the operating theater immediately after resection. Results The median electrical conductivity σ was 0.57 S/m in human colorectal liver metastases at a median temperature of 35.1℃ and 0.35 S/m in tumor-free liver tissue at a median temperature of 34.9℃. The electrical conductivity was significantly higher in tumor tissue than in tumor-free liver tissue ( P = 0.005). There were no differences in tissue temperature between the two groups ( P = 0.883). Conclusion The electrical conductivity is significantly higher in human colorectal liver metastases than in tumor-free liver tissue at a frequency of 470 kHz.

Abstract A70: Vasodilator-stimulated phosphoprotein promotes TGF-beta mediated myofibroblastic activation by regulating recycling of TGF-beta receptor II to the plasma membrane

Abstract Introduction: Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under TGF-β stimulation, hepatic stellate cells (HSCs), which are liver specific pericytes, transdifferentiate into myofibroblasts that promote tumor implantation and growth in the liver. The regulation of this HSC activation process in the liver however remains poorly understood. In this study, we tested a hypothesis that actin binding protein vasodilator-stimulated phosphoprotein (VASP), previously implicated in focal adhesion and migration of HSCs, may regulate the TGF-β mediated HSC activation process by interacting with and regulating TGF-β receptors. Methods: VASP shRNA and siRNA were used to knockdown VASP in primary human HSCs and LX2, an immortalized HSC cell line. TGF-β activation of HSCs was quantitated by immunofluorescence staining (IF) for alpha-smooth muscle actin (α-SMA) and Western blot analysis (WB) for α-SMA, fibronectin and P-SMAD2. Total TGF-β receptor levels were determined by WB, and plasma membrane TβRII was quantitated by biotinylation of cell surface proteins approach. Protein subcellular localization was investigated by IF and protein interactions were determined by double IF and co-immunoprecipitation (IP). Regulation of stellate cell VASP by HSC/tumor interactions was investigated by IF on human colorectal liver metastases and the role of stellate cell VASP in tumor growth was studied in a tumor/HSC coimplantation mouse model. Results: TGF-β1 induced more than 60% of human primary HSCs into myofibroblasts as determined by α-SMA IF. Two different VASP shRNAs significantly reduced this effect of TGF-β1 on HSC activation (P&amp;lt;0.05). The effect of VASP knockdown on HSC activation was confirmed in LX2 cells as well as using a VASP siRNA approach. VASP knockdown increased total TGF receptor protein levels with no effects on SMAD2 and 3 protein levels. VASP interacted with TβRII as evidenced by double IF and co-IP. Biotin-labeling of cell surface proteins and IF revealed that VASP knockdown reduced TβRII protein level at the plasma membrane and that VASP overexpression increased TβRII at the plasma membrane. Concurrently, VASP knockdown reduced plasma membrane localization of Rab11a, a small GTPase that marks recycling endosomes and is essential for recycling of plasma membrane receptors. VASP/Rab11 interactions were confirmed by IF and co-IP. Furthermore, we found that VASP knockdown HSCs significantly reduced tumor growth in mice as compared to control HSCs in a HSC/tumor coimplantation model. In patients, metastasis of colorectal cancer cells in liver sinusoids induced upregulation of VASP and α-SMA in adjacent HSCs as revealed by IF on biopsies of human colorectal liver metastases. Conclusions: Our studies demonstrate a requirement of VASP in the TGF-β dependent HSC activation in the tumor microenvironment and ensuing tumor growth. VASP interacts with Rab11 and regulates Rab11 positive recycling endosomal function thereby promoting TβRII recycling back to the plasma membrane in HSCs. VASP in the tumor microenvironment thus presents a therapeutic target for reducing tumor implantation and metastatic growth in the liver. Citation Format: Kangsheng Tu, Jiachu Li, Chunsheng Liu, Vijay H. Shah, Ningling Kang. Vasodilator-stimulated phosphoprotein promotes TGF-beta mediated myofibroblastic activation by regulating recycling of TGF-beta receptor II to the plasma membrane. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A70. doi:10.1158/1538-7445.CHTME14-A70

Transforming growth factor‐beta‐induced protein ig‐h3 is essential for the formation of human colorectal carcinoma liver metastasis in vivo

Transforming growth factor‐beta‐induced protein ig‐h3 (TGFBI) is extracellular matrix component important for cell‐collagen interaction. We and others have reported elevated expression of TGFBI in several human cancers, where its role remains controversial. Current study reports for the first time on the function of TGFBI in colorectal carcinoma liver metastasis (CRC‐LM), a frequently encountered malignancy with no satisfactory treatment to date.We show that TGFBI is highly expressed in human CRC‐LM and in LM originating from breast, lung and pancreatic tumors. From functional perspective, silencing TGFBI in SW1222 human colorectal carcinoma cells lead to a decrease in migration (−70%) and proliferation (−20%). To understand how this effects translate in vivo we have developed a novel animal model of colorectal carcinoma based on chicken chorioallantoic membrane (CAM) that mimics human CRC‐LM. TGFBI silencing resulted in 50% reduction of tumor volume in vivo. Finally, we have demonstrated that TGFBI is systemically reachable target for homing fluorescently‐labeled antibodies injected in tumor bearing animals. Collectively the data show that TGFBI is essential for promoting the development of CRC‐LM and therefore a promising target for designing novel therapeutic approaches.This work is supported by FNRS, Belgium and FP7 EU program.

IQGAP1 suppresses TβRII-mediated myofibroblastic activation and metastatic growth in liver.

In the tumor microenvironment, TGF-β induces transdifferentiation of quiescent pericytes and related stromal cells into myofibroblasts that promote tumor growth and metastasis. The mechanisms governing myofibroblastic activation remain poorly understood, and its role in the tumor microenvironment has not been explored. Here, we demonstrate that IQ motif containing GTPase activating protein 1 (IQGAP1) binds to TGF-β receptor II (TβRII) and suppresses TβRII-mediated signaling in pericytes to prevent myofibroblastic differentiation in the tumor microenvironment. We found that TGF-β1 recruited IQGAP1 to TβRII in hepatic stellate cells (HSCs), the resident liver pericytes. Iqgap1 knockdown inhibited the targeting of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor 1 (SMURF1) to the plasma membrane and TβRII ubiquitination and degradation. Thus, Iqgap1 knockdown stabilized TβRII and potentiated TGF-β1 transdifferentiation of pericytes into myofibroblasts in vitro. Iqgap1 deficiency in HSCs promoted myofibroblast activation, tumor implantation, and metastatic growth in mice via upregulation of paracrine signaling molecules. Additionally, we found that IQGAP1 expression was downregulated in myofibroblasts associated with human colorectal liver metastases. Taken together, our studies demonstrate that IQGAP1 in the tumor microenvironment suppresses TβRII and TGF-β dependent myofibroblastic differentiation to constrain tumor growth.

Abstract B19: IQGAP1 in the tumor microenvironment suppresses TGF-beta mediated myofibroblastic activation and ensuing tumor growth

Abstract Introduction: Transforming growth factor beta (TGF-β) within the tumor microenvironment induces transdifferentiation of quiescent pericytes and related stromal cells into myofibroblasts that promote tumor growth. However mechanisms governing TGF-β mediated myofibroblastic activation remain poorly understood. IQ motif containing GTPase activating protein 1 (IQGAP1) exerts diverse cellular functions, but its role in the tumor microenvironment is unexplored. In this study, we tested a hypothesis that IQGAP1 may interact with TGF-β receptor II (TβRII) and regulate its signaling in mesenchymal-type cells that activate into tumor associated myofibroblasts such as hepatic stellate cells (HSCs), which are resident liver pericytes, and that IQGAP1 of HSCs regulates liver metastatic growth. Experimental procedures: Retroviral and lentiviral based vectors were used to deliver TβRII, IQGAP1 or IQGAP1 shRNA into human primary HSCs. IQGAP1/TβRII interactions were determined by immunofluorescence staining, immunoprecipitation, in vitro GST pull down and in vitro protein binding assays. TGF-β1 activation of HSCs into myofibroblasts was determined by α-SMA immunofluorescence and Western Blot analyses for α-SMA, fibronectin and P-Smad2. The role of stellate cell IQGAP1 in tumorigenesis was assessed by in vitro assays of conditioned media, a HSC/tumor cell co-implantation mouse model and experimental liver metastasis model performed on IQGAP1 knockout mice. Additionally, IQGAP1 protein levels in the myofibroblasts of 29 human colorectal liver metastases were investigated by immunofluorescence staining. Results: IQGAP1 is recruited to TβRII by TGF-β1 with the C-terminal a.a. 1503-1657 region of IQGAP1 mediating IQGAP1/TβRII binding. Through scaffolding the E3 ubiquitin ligase Smurf1 and TβRII, IQGAP1 promotes ubiquitination, lysosomal and proteasomal degradation of TβRII, thus suppressing TβRII and TGF-β1 activation of HSCs into myofibroblasts in vitro. In mice, co-implantation of IQGAP1 knockdown HSCs promotes myofibroblastic activation of HSCs, tumor implantation and growth. In an experimental liver metastasis model, IQGAP1 deficiency in the tumor microenvironment promotes myofibroblastic activation and liver metastatic growth. Indeed, IQGAP1 knockdown in HSCs upregulates paracrine signaling molecules such as stromal derived factor 1 (SDF-1/CXCL12) and hepatic growth factor (HGF) that confer a stimulatory effect on proliferation, migration and survival of tumor cells. Additionally, we found that 24 out of 29 colorectal cancer patients display varying degrees of reduction of IQGAP1 protein in the myofibroblasts of their liver metastases as compared to IQGAP1 expression levels observed in activated HSCs and portal myofibroblasts of the adjacent non-tumorous control liver. Conclusion: Our studies demonstrate that IQGAP1 in the tumor microenvironment suppresses TβRII and the TGFβ1 dependent myofibroblastic activation switch with these events constraining tumor growth and metastasis. Citation Format: Chunsheng Liu, Haitham Abdelhakim, Vijay H. Shah, Ningling Kang. IQGAP1 in the tumor microenvironment suppresses TGF-beta mediated myofibroblastic activation and ensuing tumor growth. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B19.

Inhibition of the Liver Enriched Protein FOXA2 Recovers HNF6 Activity in Human Colon Carcinoma and Liver Hepatoma Cells

Recently, we demonstrated that the transcription factors HNF6 and FOXA2 function as key regulators in human colorectal liver metastases. To better understand their proposed inhibitory crosstalk, the consequences of functional knockdown of FOXA2 on HNF6 and C/EBPα activity were investigated in the human colon Caco-2 and HepG2 carcinoma cell lines. Specifically, siRNA-mediated gene silencing of FOXA2 repressed transcript expression by >80%. This resulted in a statistically significant 6-, 3-, 4-, and 8-fold increase in mRNA expression of HNF6 and of genes targeted by this transcription factor, e.g., HSP105B, CYP51, and C/EBPα, as determined by qRT-PCR. Thus, functional knockdown of FOXA2 recovered HNF6 activity. Furthermore, with nuclear extracts of Caco-2 cells no HNF6 DNA binding was observed, but expression of HNF1α, FOXA2, FOXA3, and HNF4α protein was abundant. We therefore transfected a plasmid encoding HNF6 into Caco-2 cells but also employed a retroviral vector to transfect HNF6 into HepG2 cells. This resulted in HNF6 protein expression with DNA binding activity being recovered as determined by EMSA band shift assays. Furthermore, by flow cytometry the consequences of HNF6 expression on cell cycle regulation in transfected cells was studied. Essentially, HNF6 inhibited cell cycle progression in the G2/M and G1 phase in Caco-2 and HepG2 cell lines, respectively. Here, proliferation was reduced by 80% and 50% in Caco-2 and HepG2 cells, respectively, as determined by the BrdU labeling assay. Therefore functional knockdown of FOXA2 recovered HNF6 activity and inhibited growth of tumor-cells and may possibly represent a novel therapeutic target in primary and secondary liver malignancies.

Abstract 4294: Differentiated colorectal cancer cells protect tumor-initiating cells from irinotecan

Abstract Normal colon tissue stem cells drive tissue turnover by generating daughter stem cells and a rapidly proliferating pool of progenitor cells that give rise to differentiated cell types with specialized functions. This functional hierarchy is preserved in colon tumors in which a tumor-initiating cell (or cancer stem cell) compartment drives the formation of proliferating and differentiated cell types with reduced tumorigenic potential. Normal colon stem cells possess a variety of resistance mechanisms that allow them to endure a lifetime of genotoxic insults. By inheriting these traits, tumor-initiating cells (TIC) are thought to be endowed with an intrinsic resistance to chemotherapy. However, the relationship between chemo-resistance and tumor-initiating potential in colon tumors is poorly understood. We studied this relationship using a series colonosphere cultures isolated from freshly resected primary colon tumors and liver metastases. These cultures are highly enriched for TIC, based on their high clone and tumor-forming potential and expression of the stem cell marker ALDH1. The TIC-enriched cultures display resistance to the toposomerase I inhibitor irinotecan. However, drug resistance is mediated by a small subpopulation of non-tumorigenic differentiated ALDH1-negative cells expressing the drug efflux pump ABCB1. ABCB1 is localized to the brush border of cells at the colonosphere periphery and of cells forming intra-spheroid tubules. In human colorectal liver metastases ABCB1 is predominantly localized to the luminal side of differentiated tubule-forming cells. These ABCB1 drug-resistant cells are proliferation-competent but unable to initiate tumor formation. Conversely, tumor-initiating cells are ABCB1-negative and drug-sensitive, but could generate non-tumorigenic drug-resistant offspring in vitro and in tumor xenografts. Our work shows that colorectal tumor-initiating cells do not display intrinsic resistance to irinotecan, most likely because they are derived from a normal stem cell population that does not express ABC transporters. Tumor-initiating cells generate a protective shield of drug-resistant ABCB1-positive offspring. Furthermore, the preservation of tumor-initiating capacity following irinotecan exposure requires the cooperative action of drug-sensitive tumor-initiating cells and their drug-resistant differentiated offspring. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4294.

Determination of the temperature-dependent electric conductivity of liver tissue ex vivo and in vivo: Importance for therapy planning for the radiofrequency ablation of liver tumours

Introduction: Knowledge about the changes in the electric conductivity during the coagulation process of radiofrequency ablation of the liver is a prerequisite for the predictability of produceable thermonecrosis in the liver.Materials and methods: Continuous measurements of the electric conductivity σ in ex vivo porcine liver (n = 25) were done during the coagulation and cooling process at the temperature range of the radiofrequency ablation at a frequency of 470 kHz relevant for the radiofrequency ablation. Measurements of the electric conductivity were performed in both perfused porcine liver (n = 3) and a human surgical specimen from a colorectal liver metastasis.Results: At a body temperature of 37°C, conductance σ was 0.41 siemens per metre (0.32 S/m; 0.52 S/m). Conductance σ increased continuously and uniformly at a temperature of 77°C. Maximum conductance σ with 0.79 S/m (0.7 S/m; 0.87 S/m) was reached at 80°C. A continuous reduction of conductance was observed during the cooling phase. At 37°C, the specific conductance σ in the healthy perfused porcine liver was 0.52 S/m, 0.55 S/m and 0.57 S/m (mean 0.55 S/m). The electric conductivity of the human colorectal liver metastasis was clearly higher.Conclusion: Changes in the specific conductivity during the coagulation and the cooling phase play an important role for the produceable size of a coagulation necrosis and necessitates an adaptation of the therapy parameters during radiofrequency ablation.

Stromal Fibroblasts in Colorectal Liver Metastases Originate From Resident Fibroblasts and Generate an Inflammatory Microenvironment

Cancer-associated stromal fibroblasts (CAFs) are the main cellular constituents of reactive stroma in primary and metastatic cancer. We analyzed phenotypical characteristics of CAFs from human colorectal liver metastases (CLMs) and their role in inflammation and cancer progression. CAFs displayed a vimentin(+), alpha-smooth-muscle actin(+), and Thy-1(+) phenotype similar to resident portal-located liver fibroblasts (LFs). We demonstrated that CLMs are inflammatory sites showing stromal expression of interleukin-8 (IL-8), a chemokine related to invasion and angiogenesis. In vitro analyses revealed a striking induction of IL-8 expression in CAFs and LFs by tumor necrosis factor-alpha (TNF-alpha). The effect of TNF-alpha on CAFs is inhibited by the nuclear factor-kappaB inhibitor parthenolide. Conditioned medium of CAFs and LFs similarly stimulated the migration of DLD-1, Colo-678, HuH7 carcinoma cells, and human umbilical vein endothelial cells in vitro. Pretreatment of CAFs with TNF-alpha increased the chemotaxis of Colo-678 colon carcinoma cells by conditioned medium of CAFs; however, blockage of IL-8 activity showed no inhibitory effect. In conclusion, these data raise the possibility that the majority of CAFs in CLM originate from resident LFs. TNF-alpha-induced up-regulation of IL-8 via nuclear factor-kappaB in CAFs is an inflammatory pathway, potentially permissive for cancer invasion that may represent a novel therapeutic target.

The hepatocyte nuclear factor 6 (HNF6) and FOXA2 are key regulators in colorectal liver metastases

The molecular causes leading to secondary liver malignancies are unknown. Here we report regulation of major hepatic nuclear factors in human colorectal liver metastases and primary colonic cancer. Notably, the genes coding for HNF6, HNF1beta, and C/EBPgamma were selectively regulated in liver metastases. We therefore studied protein expression of regulated transcription factors and found unacetylated HNF6 to be a hallmark of colorectal liver metastases. For its known interaction with HNF6, we investigated expression of FOXA2, which we found to be specifically induced in colorectal liver metastases. By electromobility shift assay, we examined DNA binding of disease regulated transcription factors. Essentially, no HNF6 DNA binding was observed. We also searched for sequence variations in the DNA binding domains of HNF6, but did not identify any mutation. Furthermore, we probed for expression of 28 genes targeted by HNF6. Mostly transcript expression was repressed except for tumor growth. In conclusion, we show HNF6 protein expression to be driven by the hepatic environment. Its expression is not observed in healthy colon or primary colonic cancer. HNF6 DNA binding is selectively abrogated through lack of post-translational modification and interaction with FOXA2. Targeting of FOXA2 and HNF6 may therefore enable mechanism-based therapy for colorectal liver metastases.

Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases

BackgroundThe family of proprotein convertases has been recently implicated in tumorigenesis and metastasis in animal models. However, these studies have not yet been completely corroborated in human tumors.MethodsUsing RT PCR, immunoblot and immunohistochemistry we assessed the presence and the processing patterns of the convertases PC1 and PC2 as well as the PC2 specific chaperone 7B2 in human liver metastases originating from colorectal cancer and compared them to unaffected and normal liver. Furthermore, we assessed the presence and processing profiles of PC1, PC2 and 7B2 in primary colon cancers.ResultsmRNA, protein expression, and protein cleavage profiles of proprotein convertases 1 and 2 are altered in liver colorectal metastasis, compared to unaffected and normal liver. Active PC1 protein is overexpressed in tumor, correlating with its mRNA profile. Moreover, the enhanced PC2 processing pattern in tumor correlates with the overexpression of its specific binding protein 7B2. These results were corroborated by immunohistochemistry. The specific and uniform convertase pattern observed in the metastases was present only in a fraction of primary colon cancers.ConclusionThe uniformly altered proprotein convertase profile in liver metastases is observed only in a fraction of primary colon cancers, suggesting possible selection processes involving PCs during metastasis as well as an active role of PCs in liver metastasis. In addition, the exclusive presence of 7B2 in metastatic tumors may represent a new target for early diagnosis, prognosis and/or treatment.

Comparison of Iceball Diameter and Temperature Distribution Achieved with 3-mm Accuprobe Cryoprobes in Porcine and Human Liver Tissue and Human Colorectal Liver Metastases in Vitro

We aimed to assess the thermal profile and size of iceballs produced by Accuprobe cryoprobes in fresh porcine and human liver and human colorectal cancer liver metastases in vitro to allow better planning of cryosurgical treatment of liver metastases. Iceballs were produced by a 20-min single freeze cycle using 8-mm cryoprobes in pig liver in a waterbath at 37°C (n = 8) and 3-mm cryoprobes in pig liver (n = 8), human liver (n = 3), and human colorectal cancer liver metastases (n = 8). The iceball diameters and the temperatures at different distances from the cryoprobe were measured. Mean iceball diameters produced by 8-mm cryoprobes in pig liver were 56.3 mm and varied from 38.7 to 39.6 mm for 3-mm cryoprobes in the different tissues used. There was no significant difference in iceball size in the different tissues. The diameter of the zone of −40°C or less was approximately 44 mm using 8-mm cryoprobes in porcine liver and between 27 and 31 mm using 3-mm cryoprobes in the different tissues examined. The results may allow better preoperative planning of the cryosurgical treatment of liver metastases with Accuprobe cryoprobes.

The absence of autonomic perivascular nerves in human colorectal liver metastases

The peptidergic/aminergic innervation of normal liver and tumour blood vessels was investigated in order to determine vascular control with a view to improving the efficacy of hepatic arterial cytotoxic infusion in the treatment of colorectal liver metastases. Selected areas of liver metastases and macroscopically normal liver from resection specimens (n = 13) were studied using light microscope immunohistochemistry for the presence of protein gene product 9.5 (PGP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), substance P (SP) and tyrosine hydroxylase (TH). The ultrastructure of blood vessels supplying liver metastases and their perivascular innervation were also examined by transmission electron microscopy. In the normal liver, perivascular immunoreactive nerve fibres containing PGP, NPY and TH were observed around the interlobular blood vessels and along the sinusoids and the central vein of the hepatic lobule. The greatest density of immunoreactive nerve fibres was seen for PGP, followed (in decreasing order) by NPY and TH. VIP, SP and CGRP immunoreactivity was observed only in nerve bundles associated with the large interlobular blood vessels. In contrast, no perivascular immunoreactive nerves were observed in colorectal liver metastases. Electron microscopy confirmed the absence of perivascular nerves in liver metastases. In addition, it showed that the walls of these blood vessels were composed of a layer of endothelial cells surrounded by an incomplete or, very rarely in the periphery of the tumour, a complete, layer of synthetic phenotype of smooth muscle-like cells. These results imply that the blood vessels supplying liver metastases are bereft of normal neuronal regulation; whether there is a role for endothelial cell control of blood flow in these vessels is not yet known.