Roni F. Rayes

Neutrophil extracellular traps sequester circulating tumor cells via β1-integrin mediated interactions

Despite advances in cancer treatment, metastasis remains today the main cause of cancer death. Local control through complete surgical resection of the primary tumor continues to be a key principle in cancer treatment. However, surgical interventions themselves lead to adverse oncologic outcomes and are associated with significantly increased rates of metastasis. Neutrophils through release of neutrophil extracellular traps (NETs) in response to infections were shown to be able to capture circulating cancer cells, and in doing so, support the development of metastatic disease. To be able to intervene on this process, understanding the exact molecular nature of these mechanisms is crucial. We therefore hypothesize and demonstrate that β1‐integrin is an important factor mediating the interactions between circulating tumor cells and NETs. We show that β1‐integrin expression on both cancer cells and NETs is important for the adhesion of circulating tumor cells to NETs both in vitro and in vivo. Using a murine model of intra‐abdominal sepsis to mimic the postoperative inflammatory environment, we show that β1‐integrin expression is upregulated in the context of inflammation in vivo. Ultimately, we show that this increased early cancer cell adhesion to NETs in vivo and this effect is abrogated when mice are administered DNAse 1. Our data therefore sheds light on the first molecular mechanism by which NETs can trap circulating tumor cells (CTCs), broadening our understanding of this process.

The IGF-Trap: Novel Inhibitor of Carcinoma Growth and Metastasis

The IGFI receptor promotes malignant progression and has been recognized as a target for cancer therapy. Clinical trials with anti-IGFIR antibodies provided evidence of therapeutic efficacy but exposed limitations due in part to effects on, and the compensatory function of, the insulin receptor system. Here, we report on the production, characterization, and biologic activity of a novel, IGF-targeting protein (the IGF-Trap) comprising a soluble form of hIGFIR and the Fc portion of hIgG1. The IGF-Trap has a high affinity for hIGFI and hIGFII but low affinity for insulin, as revealed by surface plasmon resonance. It efficiently blocked IGFIR signaling in several carcinoma cell types and inhibited tumor cell proliferation, migration, and invasion in vitro. In vivo, the IGF-Trap showed favorable pharmacokinetic properties and could suppress the growth of established breast carcinoma tumors when administered therapeutically into tumor-bearing mice, improving disease-free survival. Moreover, IGF-Trap treatment markedly reduced experimental liver metastasis of colon and lung carcinoma cells, increasing tumor cell apoptosis and reducing angiogenesis. Finally, when compared with an anti-IGFIR antibody or IGF-binding protein-1 that were used at similar or higher concentrations, the IGF-Trap showed superior therapeutic efficacy to both inhibitors. Taken together, we have developed a targeted therapeutic molecule with highly potent anticancer effects that could address limitations of current IGFIR-targeting agents. Mol Cancer Ther; 14(4); 982–93. ©2015 AACR.

International consensus guidelines for scoring the histopathological growth patterns of liver metastasis

Background:Liver metastases present with distinct histopathological growth patterns (HGPs), including the desmoplastic, pushing and replacement HGPs and two rarer HGPs. The HGPs are defined owing to the distinct interface between the cancer cells and the adjacent normal liver parenchyma that is present in each pattern and can be scored from standard haematoxylin-and-eosin-stained (H&E) tissue sections. The current study provides consensus guidelines for scoring these HGPs.Methods:Guidelines for defining the HGPs were established by a large international team. To assess the validity of these guidelines, 12 independent observers scored a set of 159 liver metastases and interobserver variability was measured. In an independent cohort of 374 patients with colorectal liver metastases (CRCLM), the impact of HGPs on overall survival after hepatectomy was determined.Results:Good-to-excellent correlations (intraclass correlation coefficient >0.5) with the gold standard were obtained for the assessment of the replacement HGP and desmoplastic HGP. Overall survival was significantly superior in the desmoplastic HGP subgroup compared with the replacement or pushing HGP subgroup (P=0.006).Conclusions:The current guidelines allow for reproducible determination of liver metastasis HGPs. As HGPs impact overall survival after surgery for CRCLM, they may serve as a novel biomarker for individualised therapies.

The type I insulin-like growth factor regulates the liver stromal response to metastatic colon carcinoma cells

Hepatic stellate cells (HSC) play a major role in initiating the liver fibrogenic (wounding) response of the liver and can also orchestrate a pro-metastatic microenvironment in the liver in response to invading cancer cells. Here we explored the role of the hepatic stellate cells in colon carcinoma liver metastasis with emphasis on the contribution of the insulin-like growth factor (IGF) axis to their activation and function. To this end, we used mice with a Tamoxifen inducible liver IGF-I deficiency. We found that in mice with a sustained IGF-I deficiency, recruitment and activation of HSC into tumor-infiltrated areas of the liver were markedly diminished, resulting in decreased collagen deposition and reduced tumor expansion. In addition, IGF-I could rescue HSC from apoptosis induced by pro-inflammatory factors such as TNF-α known to be upregulated in the early stages of liver metastasis. Moreover, in surgical specimens, activated IGF-IR was observed on HSC-like stromal cells surrounding colorectal carcinoma liver metastases. Finally, IGF-targeting in vivo using an IGF-Trap caused a significant reduction in HSC activation in response to metastatic colon cancer cells. Therefore, our data identify IGF as a survival factor for HSC and thereby, a promoter of the pro-metastatic microenvironment in the liver. IGF-targeting could therefore provide a strategy for curtailing the pro-metastatic host response of the liver during the early stages of liver metastasis.Hepatic stellate cells (HSC) play a major role in initiating the liver fibrogenic (wounding) response of the liver and can also orchestrate a pro-metastatic microenvironment in the liver in response to invading cancer cells. Here we explored the role of the hepatic stellate cells in colon carcinoma liver metastasis with emphasis on the contribution of the insulin-like growth factor (IGF) axis to their activation and function. To this end, we used mice with a Tamoxifen inducible liver IGF-I deficiency. We found that in mice with a sustained IGF-I deficiency, recruitment and activation of HSC into tumor-infiltrated areas of the liver were markedly diminished, resulting in decreased collagen deposition and reduced tumor expansion. In addition, IGF-I could rescue HSC from apoptosis induced by pro-inflammatory factors such as TNF-α known to be upregulated in the early stages of liver metastasis. Moreover, in surgical specimens, activated IGF-IR was observed on HSC-like stromal cells surrounding colorectal carcinoma liver metastases. Finally, IGF-targeting in vivo using an IGF-Trap caused a significant reduction in HSC activation in response to metastatic colon cancer cells. Therefore, our data identify IGF as a survival factor for HSC and thereby, a promoter of the pro-metastatic microenvironment in the liver. IGF-targeting could therefore provide a strategy for curtailing the pro-metastatic host response of the liver during the early stages of liver metastasis.

Loss of neutrophil polarization in colon carcinoma liver metastases of mice with an inducible, liver-specific IGF-I deficiency

The growth of cancer metastases in the liver depends on a permissive interaction with the hepatic microenvironment and neutrophils can contribute to this interaction, either positively or negatively, depending on their phenotype. Here we investigated the role of IGF-I in the control of the tumor microenvironment in the liver, using mice with a conditional, liver-specific, IGF-I deficiency (iLID) induced by a single tamoxifen injection. In mice that had a sustained (3 weeks) IGF-I deficiency prior to the intrasplenic/portal inoculation of colon carcinoma MC-38 cells, we observed an increase in neutrophil accumulation in the liver relative to controls. However, unlike controls, these neutrophils did not acquire the (anti-inflammatory) tumor-promoting phenotype, as evidenced by retention of high ICAM-1 expression and nitric oxide production and low CXCR4, CCL5, and VEGF expression and arginase production, all characteristic of the (pro-inflammatory) phenotype. This coincided with an increase in apoptotic tumor cells and reduced metastasis. Neutrophils isolated from these mice also had reduced IGF-IR expression levels. These changes were not observed in iLID mice with a short-term (2 days) IGF-I depletion, despite a 70% reduction in their circulating IGF-I levels, indicating that a sustained IGF-I deficiency was necessary to alter the neutrophil phenotype. Similar results were obtained with the highly metastatic Lewis lung carcinoma subline H-59 cells and in mice injected with an IGF-Trap that blocks IGF-IR signaling by reducing ligand bioavailability. Our results implicate the IGF axis in neutrophil polarization and the induction of a pro-metastatic microenvironment in the liver.

Collagen IV-conveyed signals can regulate chemokine production and promote liver metastasis

Liver metastases remain a major cause of death from gastrointestinal tract cancers as well as from other malignancies such as breast and lung carcinomas and melanoma. Understanding the underlying biology is essential for the design of effective targeted therapies. We previously reported that collagen IV α1/α2 overexpression in non-metastatic lung carcinoma (M27colIV) cells increased their metastatic ability, specifically to the liver and documented high collagen IV levels in surgical resections of liver metastases from diverse tumor types. Here, we aimed to elucidate the functional relevance of collagen IV to metastatic outgrowth in the liver. Gene expression profiling revealed in M27colIVcells significant increases in the expression of chemokines CCL5 (5.7-fold) and CCL7 (2.6-fold) relative to wild-type cells, and this was validated by qPCR and western blotting. Similarly, in human colon carcinoma KM12C and KM12SM cells with divergent liver-colonizing potentials, CCL7 and CCL5 production correlated with type IV collagen expression and the metastatic phenotype. CCL7 silencing by short hairpin RNA (shRNA) reduced experimental liver metastasis in both cell types, whereas CCL5 silencing reduced metastasis of M27colIV cells, implicating these cytokines in metastatic expansion in the liver. Subsequent functional analyses implicated both MEK/ERK and PI3K signaling upstream of CCL7 upregulation and identified CCL7 (but not CCL5) as a critical migration/invasion factor, acting via the chemokine receptor CCR3. Chemokine CCL5 was identified as a regulator of the T-cell immune response in the liver. Loss of CCL7 in KM12SM cells was also associated with altered E-cadherin and reduced vimentin and Snail expression, implicating it in epithelial-to-mesenchymal transition in these cells. Moreover, in clinical specimens of colon cancer liver metastases analyzed by immunohistochemistry, CCL5 and CCL7 levels paralleled those of collagen IV. The results identify the chemokines CCL5 and CCL7 as type IV collagen-regulated genes that promote liver metastasis by distinct and complementary mechanisms.

Abstract 5081: The IGF axis regulates hepatic stellate cell recruitment and activation during colorectal carcinoma liver metastasis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: The hepatic stellate cells (HSC) play a major role in orchestrating the livers fibrogenic (wounding) response and have been identified as an important player in the prometastatic microenvironment in the liver. Here we analyzed the role of the IGF axis in the recruitment and activation of HSC during the early stages of colorectal carcinoma (CRC) liver metastasis. Methods: Murine CRC MC-38 cells were inoculated via the intrasplenic/portal route into female mice with a conditional liver IGF-1 deletion (iLID), induced by a single tamoxifen (Tx) injection 2 days or 3 weeks earlier. Vehicle (sunflower oil) injected iLID mice or Tx - injected wild type mice were used as controls. Experimental liver fibrosis was achieved by repeated carbon tetrachloride (CCl4) administration. The stromal response of the liver was analyzed using immunohistochemistry with emphasis on HSC recruitment and activation. In addition, in vitro assays were utilized to explore the role of IGF-1 in HSC activation. Results: In iLID mice treated with Tx, a 75% reduction in circulating IGF-1 levels could be observed within 24 hr and it persisted for the duration of the experiments. When injected with MC-38 cells, 3 weeks post Tx injection, these mice developed significantly fewer liver metastases than non-treated controls, while no reduction in the number of metastases was seen in WT mice injected with Tx or in oil-injected iLID controls. Interestingly, we observed that Tx treatment 48 hr prior to tumor injection, failed to reduce liver metastasis in iLID mice, although their circulating IGF-1 levels were markedly reduced, suggesting that the loss of direct paracrine IGF-1 effects on the tumor cells was not sufficient to inhibit tumor growth in the liver and that other effects on the hepatic microenvironment were at play. Analysis of HSC recruitment and activation subsequently revealed a significant reduction in HSC activation around micrometastases as compared to controls. This was evident as early as 3 days post tumor inoculation, persisted for the duration of the experimental period (16-18 days) and corresponded with reduced IGF-1 receptor and Akt activation in these cells. In vitro studies confirmed that IGF-1 could directly activate isolated HSC and rescue them from serum-depletion induced apoptosis. Finally, in iLID mice with sustained IGF-1 depletion, a significant reduction in HSC-mediated collagen deposition was observed following continuous treatment with CCl4, confirming the role of IGF-1 in HSC activation in a second tumor-free model. Conclusion: Our results show that a sustained reduction in circulating IGF-1 levels altered HSC recruitment and activation to tumor sites and reduced tumor cell growth in the liver. We identify IGF-1 as a regulator of HSC function and the response of the microenvironment to invading cancer cells, thereby affecting metastatic expansion. Supported by CIHR Grant MOP 81201 (to PB) and MICRTP Fellowship (to MCF). Note: This abstract was not presented at the meeting. Citation Format: Maria C. Fernandez, Roni F. Rayes, Jun Xu, Tatiana Kisseleva, Shoshana Yakar, Pnina Brodt. The IGF axis regulates hepatic stellate cell recruitment and activation during colorectal carcinoma liver metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5081. doi:10.1158/1538-7445.AM2015-5081

Abstract 2094: Regulation of site-specific liver metastasis by collagen IV-conveyed signals

The liver is a primary site of metastasis for some of the most common human malignancies. At present, surgical resection is the most effective curative option for liver metastases, but most patients with liver metastases still succumb to their disease. A better understanding of the underlying biology is essential for design of more effective therapy. We previously identified basement membrane type IV collagen α1/α2 expression levels as determinants of the liver colonizing potential in a murine lung carcinoma model (1) and observed high collagen IV expression levels in surgical specimens of liver metastases from diverse tumor types. The aim of this study was to elucidate the functional relevance of increased collagen IV expression to liver metastases formation. We reported that collagen IV α1/α2 overexpression in non-metastatic lung carcinoma M-27 (M27colIV) cells increased specifically their liver (but not lung) metastasizing potential. A transcriptome analysis was therefore performed on these cells in order to identify changes to gene expression that could account for the newly acquired metastatic potential. This analysis revealed that type IV collagen levels regulated the expression of multiple genes. Prominent among them were genes encoding for chemokines such as CCL-5 and CCL-7 and for growth factors such as amphiregulin (AREG) that were all upregulated by ≥ 3 fold. These changes were validated at the RNA and protein levels using qPCR and Western blotting, respectively. When the expression of these genes in M27colIV cells was subsequently silenced using shRNA, a significant decrease (5-10 folds) was observed in their ability to generate experimental liver metastases, as compared to wild type or mock-transfected cells and they failed to develop metastases in 40-50 % of injected mice. A similar correlation between CCL-5 and CCL-7 expression levels and liver metastases formation was also noted in human colon carcinoma cell lines. The results identify a type IV collagen-regulated gene expression signature that promotes liver metastasis and suggest that collagen IV-induced changes in chemokine and growth factor production levels mediate this effect. They provide a rationale for further exploration of the clinical utility of CCL-5, CCL-7 and AREG as targets in the management of liver metastases. Supported by a grant from the Canadian Institute for Health Research (to PB) and a Henry R. Shibata Cedars Cancer Fellowship (to RR). REFERENCES 1. Burnier JV, Wang N, Michel RP, Hassanain M, Li S, Lu Y, et al. Type IV collagen-initiated signals provide survival and growth cues required for liver metastasis. Oncogene. 2011;30:3766-83. Citation Format: Roni F. Rayes, Ni Wang, Julia V. Burnier, France Bourdeau, Pnina Brodt. Regulation of site-specific liver metastasis by collagen IV-conveyed signals. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2094. doi:10.1158/1538-7445.AM2014-2094