Pro-metastatic Microenvironment Research Articles

Novel View on Unwanted Side Effects of Radio-Chemotherapy on Bone Marrow (BM) Microenvironment - Radio-Chemotherapy Upregulates BM-Level of Bioactive Lipids, Sphingosine-1- Phosphate (S1P) and Ceramide-1-Phosphate (C1P), That Chemoattract Metastasizing Cancer Cells

Abstract 726 Background:Rhabdomyosracoma (RMS), the most common soft-tissue sarcoma of adolescents and children, frequently infiltrates the BM to such a degree that it often mimics acute lymphoblastic leukemia. The prognosis is poor, particularly for the more aggressive and metastatic alveolar RMS (ARMS) compared to embryonal RMS (ERMS). In our previous work, we demonstrated a pivotal role for two signaling axes, a-chemokine stromal-derived factor-1 (SDF-1)–CXCR4 (a seven-transmembrane-spanning G protein-coupled receptor) and hepatocyte growth factor/scatter factor (HGF/SF)–c-met, in metastasis of pediatric sarcomas to bone marrow (BM) (Blood 2002;100:2597-2606,Cancer Research 2003; 63:7926–7935, IJC 2010;127: 2554–2568). Recently, however, we observed that the bioactive lipids sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are much more potent chemotractants for human rhabdomyosarcoma (RMS) than SDF-1 or HGF/SF. Importantly, we observed that S1P and C1P levels are highly increased in BM after radio-chemotherapy. Hypothesis: Based on these observations, we hypothesized that S1P and C1P direct chemotaxis of RMS cells to BM. This could be particularly important in patients treated with radio-chemotherapy, where upregulation of S1P and C1P levels in BM may facilitate the spread to the bones of tumor cells that survived initial treatment. Material and Methods: Several complementary in vitro and in vivo approaches were employed to demonstrate a novel role of bioactive lipids in BM metastasis of RMS cells. The expression of S1P seven-transmembrane-spanning G protein-coupled receptors, chemotaxis, adhesion, proliferation, and cell signaling studies in response to S1P and C1P were performed on 8 human ARMS and 3 human ERMS cell lines. The secretion of S1P and C1P in BM and by RMS cells was measured by mass spectrometry (MS). The S1P1 receptor was downregulated by employing an shRNA strategy and S1P1-KO cells were evaluated for their ability to grow tumors in immunodeficient mice. Finally, to address the role of the S1 P–S 1P1 axis in the unwanted spread of sarcoma cells after radio-chemotherapy, we compared seeding of S1P1-KO and control RMS cells in irradiated immunodeficient mice. Results: S1P and C1P are much more potent chemoattractants than SDF-1 or HGF/SF, particularly if employed at “physiological” tissue concentrations. S1P1–5 receptors are expressed on RMS cells and stimulation by S1P induced chemotaxis, adhesion of these cells, and phosphorylation of MAPPp42/44 and AKT. However, while receptor/s for C1P have not yet been identified, C1P also exerted similar effects on human RMS cells. Finally, S1P1-KO cells grew smaller tumors in immunodefcient mice and had impaired seeding efficiency in irradiated animals compared to control RMS cells transduced with empty vector. In parallel experiments, we also observed that both bioactive lipids increase stromalization of the RMS by i) chemoattracting and activating cancer-associated fibroblasts (CAF) and ii) promoting tumor angiogenesis. Conclusions: Both systemic and local radio-chemotherapy leads to upregulation of bioactive lipids in damaged tissues and side effect of such treatment is induction of unwanted prometastatic microenvironment in different organs. By employing an RMS model, we confirmed S1P and identified C1P as novel under-appreciated factor directing metastasis of cancer cells. Since S1P and C1P become upregulated in BM after radio-chemotherapy, both bioactive lipids are involved in the unwanted spread to the bones of RMS cells that survived initial treatment. The role of S1P and C1P in metastasis of other pediatric sarcomas and other types of solid tumors and dissemination of leukemias/lymphomas is currently being investigated in our laboratory, similarly as different strategies to inhibit pro-metastatic effects of S1P and C1P. Disclosures:No relevant conflicts of interest to declare.

Abstract B96: Hepatic microenvironment-dependent colon cancer genes: New targets for antimetastatic therapy.

Abstract The liver is the first organ where colon cancers deliver their soluble molecules and circulating cancer cells. The effect of colon cancer products results in the “liver prometastatic reaction”, a special microenvironment supporting the experimental hepatic colonization process of colon cancer cells. However, at present it is unclear which genes tumor-activated hepatic cells specifically regulate in colon cancer cells for supporting their intrahepatic growth. We used a microenvironmental approach to the study of genes associated with colon cancer cells' ability to metastasize to the liver in patients with advanced colon cancer. First we determined by cDNA microarrays and reverse transcription-PCR hepatic metastasis genes not expressed in tumor-unaffected areas of the same liver but expressed in the primary tumors of patients that developed metastases within 5 years of diagnosis. Among identified hepatic metastasis genes, we next selected those that overlapped with genes whose expression level changed in HT-29 human colon carcinoma cells given the conditioned medium from tumor-activated primary cultured human hepatocytes and hepatic stellate cell-derived myofibroblasts. Second, we determined genes co-expressed in hepatic metastases and tumor-unaffected liver tissue, but neither in the primary tumors nor in normal liver. From this gene category we selected those expressed either by cultured HT-29 cells treated with liver cell-conditioned media, or by primary cultured liver cells receiving HT-29-conditioned media. Identified genes suggested that metastatic colon cancer have hepatomimetic properties, and that hepatic tissue expressed colon cancer specific genes under the regulation of tumor-derived factors. A third approach was to determine genes of tumor unaffected areas from livers bearing colon cancer metastasis, not expressed by colon cancer cells. This gene category represented the genetic background of the liver prometastatic reaction induced by colon cancer derived factors, and therefore, genes at the colon cancer metastasis microenvironment. We identified microenvironment-related colon cancer genes whose expression level was regulated by hepatocytes, hepatic myofibroblasts or both. We also identified some genes representing the liver prometastatic reaction whose specific role in the pathogenesis of hepatic metastasis has already been reported. Identified genes may provide a new target context for therapeutic innovation in the cancer metastasis microenvironment of colon cancer patients. On the one hand, by developing drugs able to block liver factors supporting colon cancer metastasis genes and therefore preventing effects of the “liver prometastatic reaction” in colon cancer patients. On the other hand, by developing drugs able to block those factors from colon cancer cells able to induce the prometastatic microenvironment of the liver. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B96.

Discoidin domain receptor 2 deficiency predisposes hepatic tissue to colon carcinoma metastasis

BackgroundThe transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts is a major mechanism for stroma development in hepatic metastasis, but their regulatory pathways remain unclear. Transdifferentiated HSCs from fibrotic liver...

The Liver Prometastatic Reaction of Cancer Patients: Implications for Microenvironment-Dependent Colon Cancer Gene Regulation

Colon cancer frequently metastasizes to the liver but the genetic and phenotypic properties of specific cancer cells able to implant and grow in this organ have not yet been established. The contribution of the patient's genetic, physiologic and pathologic backgrounds to the incidence and development of hepatic colon cancer metastases is also presently misunderstood. At a transcriptional level, hepatic metastasis development is in part associated with marked changes in gene expression of colon cancer cells that may originate in the primary tumor. Other changes occur in the liver and are regulated by hepatic cells, which represent the new microenvironment for metastatic colon cancer cells. However, hepatic parenchymal and non-parenchymal cell functions are also affected by both tumor-derived factors and systemic host factors, which suggests that the hepatic metastasis microenvironment is a functional linkage between the hepatic pathophysiology of the colon cancer patient and the biology of its cancer cells. Therefore, together with metastasis-related gene profiles suggesting the existence of liver metastasis potential in primary tumors, new biomarkers of the prometastatic microenvironment supported by the liver reaction to colon cancer factors may be helpful for the individual assessment of hepatic metastasis risk in colon cancer patients. In addition, knowledge on hepatic metastasis gene regulation by the hepatic microenvironment may open multiple opportunities for therapeutic intervention during colon cancer metastasis at both subclinical and advanced stages.

Hepatic stellate cells: Partners in crime for liver metastases?

Hepatic stellate cells (HSCs) were recently postulated as a component of the prometastatic liver microenvironment, because they can transdifferentiate into highly proliferative and motile myofibroblasts that are implicated in the desmoplastic reaction and metastatic growth. This review focuses on bidirectional interactions between tumor cells and HSCs in the liver microenvironment and discusses mechanisms whereby tumor-derived factors activate HSCs, and in turn, activated HSCs promote metastatic growth. Bidirectional interactions between tumors and HSCs may function as an "amplification loop" to further enhance metastatic growth in the liver. The activation of HSCs is a complex process regulated by multiple factors such as transforming growth factor-β and platelet-derived growth factor signaling pathways, which may present as therapeutic targets in the prevention and treatment of liver metastases. HSCs may present a new therapeutic target in the treatment of liver metastases. Targeting HSCs and/or myofibroblasts with transforming growth factor-β or platelet-derived growth factor antagonists in coordination with chemotherapy, radiotherapy, or surgery may prove to be effective at reducing liver metastases and increasing the survival benefit of patients by targeting both tumor cells and the tumor microenvironment.

Resveratrol prevents inflammation-dependent hepatic melanoma metastasis by inhibiting the secretion and effects of interleukin-18

BackgroundImplantation and growth of metastatic cancer cells at distant organs is promoted by inflammation-dependent mechanisms. A hepatic melanoma metastasis model where a majority of metastases are generated via interleukin-18-dependent mechanisms was used to test whether anti-inflammatory properties of resveratrol can interfere with mechanisms of metastasis.MethodsTwo experimental treatment schedules were used: 1) Mice received one daily oral dose of 1 mg/kg resveratrol after cancer cell injection and the metastasis number and volume were determined on day 12. 2) Mice received one daily oral dose of 1 mg/kg resveratrol along the 5 days prior to the injection of cancer cells and both interleukin-18 (IL-18) concentration in the hepatic blood and microvascular retention of luciferase-transfected B16M cells were determined on the 18th hour. In vitro, primary cultured hepatic sinusoidal endothelial cells were treated with B16M-conditioned medium to mimic their in vivo activation by tumor-derived factors and the effect of resveratrol on IL-18 secretion, on vascular cell adhesion molecule-1 (VCAM-1) expression and on tumor cell adhesion were studied. The effect of resveratrol on melanoma cell activation by IL-18 was also studied.ResultsResveratrol remarkably inhibited hepatic retention and metastatic growth of melanoma cells by 50% and 75%, respectively. The mechanism involved IL-18 blockade at three levels: First, resveratrol prevented IL-18 augmentation in the blood of melanoma cell-infiltrated livers. Second, resveratrol inhibited IL-18-dependent expression of VCAM-1 by tumor-activated hepatic sinusoidal endothelium, preventing melanoma cell adhesion to the microvasculature. Third, resveratrol inhibited adhesion- and proliferation-stimulating effects of IL-18 on metastatic melanoma cells through hydrogen peroxide-dependent nuclear factor-kappaB translocation blockade on these cells.ConclusionsThese results demonstrate multiple sites for therapeutic intervention using resveratrol within the prometastatic microenvironment generated by tumor-induced hepatic IL-18, and suggest a remarkable effect of resveratrol in the prevention of inflammation-dependent melanoma metastasis in the liver.

Tissue inhibitor of metalloproteinases‐1‐induced scattered liver metastasis is mediated by host‐derived urokinase‐type plasminogen activator

Paradoxically, not only proteinases but also their inhibitors can correlate with bad prognosis of cancer patients, underlining the evolving concept of the protease web as the complex interplay between proteinases, their inhibitors and effector molecules. Elevated levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) render the liver more susceptible to metastasis by triggering urokinase plasminogen activator (uPA) expression as well as hepatocyte growth factor (HGF) signalling, thereby leading to the fatal scattered infiltration of metastasizing tumour cells throughout the parenchyma of the target organ. Here, we investigated whether host uPA is a crucial protagonist for the TIMP-1-induced modulation of a pro-metastatic microenvironment in the liver. Indeed, in livers of uPA-ablated mice elevated TIMP-1 levels did not trigger HGF signalling and did not promote metastasis of a murine T-lymphoma cell line. In contrast, lack of tumour cell-derived uPA induced by gene silencing did not interfere with this pro-metastatic pathway. Furthermore, host uPA was necessary for the recruitment of neutrophilic granulocytes and the associated increase of HGF in livers with elevated TIMP-1 levels. This newly identified co-operation between TIMP-1 and host uPA suggests that therapies, simultaneously interfering with pro- and anti-proteolytic pathways may be beneficial for patients with metastatic disease.

TNF-α similarly induces IL-6 and MCP-1 in fibroblasts from colorectal liver metastases and normal liver fibroblasts

Cancer-associated fibroblasts (CAFs) represent the predominant cell type of the neoplastic stroma of solid tumors, yet their biology and functional specificity for cancer pathogenesis remain unclear. We show here that primary CAFs from colorectal liver metastases express several inflammatory, tumor-enhancing factors, including interleukin (IL)-6 and monocyte-chemoattractant protein (MCP)-1. Both molecules were intensely induced by TNF-α on the transcript and protein level, whereas PDGF-BB, TGF-β1 and EGF showed no significant effects. To verify their potential specialization for metastasis progression, CAFs were compared to fibroblasts from non-tumor liver tissue. Interestingly, these liver fibroblasts (LFs) displayed similar functions. Further analyses revealed a comparable up-regulation of intercellular adhesion molecule-1 (ICAM-1) by TNF-α, and of alpha-smooth muscle actin, by TGF-β1. Moreover, the proliferation of both cell types was induced by PDGF-BB, and CAFs and LFs displayed an equivalent migration towards HT29 colon cancer cells in Boyden chamber assays. In conclusion, colorectal liver metastasis may be supported by CAFs and resident fibroblastic cells competent to generate a prometastatic microenvironment through inflammatory activation of IL-6 and MCP-1.

Lack of host SPARC enhances vascular function and tumor spread in an orthotopic murine model of pancreatic carcinoma

Utilizing subcutaneous tumor models, we previously validated SPARC (secreted protein acidic and rich in cysteine) as a key component of the stromal response, where it regulated tumor size, angiogenesis and extracellular matrix deposition. In the present study, we demonstrate that pancreatic tumors grown orthotopically in Sparc-null (Sparc(-/-)) mice are more metastatic than tumors grown in wild-type (Sparc(+/+)) littermates. Tumors grown in Sparc(-/-) mice display reduced deposition of fibrillar collagens I and III, basement membrane collagen IV and the collagen-associated proteoglycan decorin. In addition, microvessel density and pericyte recruitment are reduced in tumors grown in the absence of host SPARC. However, tumors from Sparc(-/-) mice display increased permeability and perfusion, and a subsequent decrease in hypoxia. Finally, we found that tumors grown in the absence of host SPARC exhibit an increase in alternatively activated macrophages. These results suggest that increased tumor burden in the absence of host SPARC is a consequence of reduced collagen deposition, a disrupted vascular basement membrane, enhanced vascular function and an immune-tolerant, pro-metastatic microenvironment.

The Prometastatic Microenvironment of the Liver

The liver is a major metastasis-susceptible site and majority of patients with hepatic metastasis die from the disease in the absence of efficient treatments. The intrahepatic circulation and microvascular arrest of cancer cells trigger a local inflammatory reaction leading to cancer cell apoptosis and cytotoxicity via oxidative stress mediators (mainly nitric oxide and hydrogen peroxide) and hepatic natural killer cells. However, certain cancer cells that resist or even deactivate these anti-tumoral defense mechanisms still can adhere to endothelial cells of the hepatic microvasculature through proinflammatory cytokine-mediated mechanisms. During their temporary residence, some of these cancer cells ignore growth-inhibitory factors while respond to proliferation-stimulating factors released from tumor-activated hepatocytes and sinusoidal cells. This leads to avascular micrometastasis generation in periportal areas of hepatic lobules. Hepatocytes and myofibroblasts derived from portal tracts and activated hepatic stellate cells are next recruited into some of these avascular micrometastases. These create a private microenvironment that supports their development through the specific release of both proangiogenic factors and cancer cell invasion- and proliferation-stimulating factors. Moreover, both soluble factors from tumor-activated hepatocytes and myofibroblasts also contribute to the regulation of metastatic cancer cell genes. Therefore, the liver offers a prometastatic microenvironment to circulating cancer cells that supports metastasis development. The ability to resist anti-tumor hepatic defense and to take advantage of hepatic cell-derived factors are key phenotypic properties of liver-metastasizing cancer cells. Knowledge on hepatic metastasis regulation by microenvironment opens multiple opportunities for metastasis inhibition at both subclinical and advanced stages. In addition, together with metastasis-related gene profiles revealing the existence of liver metastasis potential in primary tumors, new biomarkers on the prometastatic microenvironment of the liver may be helpful for the individual assessment of hepatic metastasis risk in cancer patients.

Inhibition of cytokine-induced microvascular arrest of tumor cells by recombinant endostatin prevents experimental hepatic melanoma metastasis.

We investigated effects of endostatin (ES) in the prometastatic microenvironment of inflammation occurring during the microvascular phase of cancer cell infiltration in the liver. We used a model of intrasplenic injection of B16 melanoma (B16M) cells leading to hepatic metastasis through vascular cell adhesion molecule-(VCAM-1)-mediated capillary arrest of cancer cells via interleukin-18 (IL-18)-dependent mechanism. We show that administration of 50 mg/kg recombinant human (rh) ES 30 min before B16M, plus repetition of same dose for 3 additional days decreased metastasis number by 60%. A single dose of rhES before B16M injection reduced hepatic microvascular retention of luciferase-transfected B16M by 40% and inhibited hepatic production of tumor necrosis factor alpha (TNF-alpha) and IL-18 and VCAM-1 expression by hepatic sinusoidal endothelia (HSE). Consistent with these data, rhES inhibited VCAM-1-dependent B16M cell adhesion to primary cultured HSE receiving B16M conditioned medium, and it abolished the HSE cell production of TNF-alpha and IL-18 induced by tumor-derived vascular endothelial cell growth factor (VEGF). rhES abrogated recombinant murine VEGF-induced tyrosine phosphorylation of KDR/flk-1 receptor in HSE cells, preventing the proinflammatory action of tumor-derived VEGF on HSE. rhES also abolished hepatic production of TNF-alpha, microvascular retention of luciferase-transfected B16M, and adhesion of B16M cells to isolated HSE cells, all of them induced in mice given 5 micro g/kg recombinant murine VEGF for 18 h. This capillary inflammation-deactivating capability constitutes a nonantiangiogenic antitumoral action of endostatin that decreases cancer cell arrest within liver microvasculature and prevents metastases promoted by proinflammatory cytokines induced by VEGF.

Hydrogen peroxide mediates vascular cell adhesion molecule-1 expression from interleukin-18–activated hepatic sinusoidal endothelium: Implications for circulating cancer cell arrest in the murine liver

The mechanism of intrasinusoidal arrest of circulating cancer cells, which is a critical step in liver metastasis, appears to be facilitated by tumor-derived proinflammatory factors that increase sinusoidal cell adhesion receptors for cancer cells. However, how this prometastatic microenvironment is up-regulated remains unknown. Using intrasplenically injected B16 melanoma (B16M) cells, we show that the expression of vascular cell adhesion molecule-1 (VCAM-1) significantly increased in hepatic sinusoidal endothelium (HSE) cells over physiologic baseline within the first 24 hours of metastatic cancer cell infiltration in the liver. This correlated with increased in vitro adhesion of B16M cells to HSE cells isolated from B16M cell–injected mice. In vivo VCAM-1 blockade with specific antibodies before B16M cell injection decreased sinusoidal retention of luciferase-transfected B16M cells by 85%, and metastasis development by 75%, indicating that VCAM-1 expression on tumor-activated HSE cells had a prometastatic contribution. Because VCAM-1 expression is oxidative stress–inducible, recombinant catalase was in vivo administered, resulting in a complete abrogation of both VCAM-1 expression and B16M cell adhesion increases in HSE cells isolated from B16M cell–injected mice. Catalase also abrogated the proadhesive response of HSE cells to B16M-conditioned medium (B16M-CM) in vitro, although this did not affect the concomitant release of major proinflammatory cytokines by HSE cells. HSE cells treated with B16M-CM released interleukin (IL)-18 via tumor necrosis factor-α (TNF-α)–dependent IL-1β in vitro. In turn, H2O2 production from B16M-CM–treated HSE cells was regulated by IL-18. Thus, liver-infiltrating B16M cells activated their adhesion to HSE through a sequential process involving TNF-α–dependent IL-1β, which induced IL-18 to up-regulate VCAM-1 via H2O2. The pivotal position of H2O2 was further supported by the fact that incubation of HSE cells with nontoxic concentrations of H2O2 directly enhanced VCAM-1–dependent B16M cell adhesion in vitro without proinflammatory cytokine mediation, which emphasizes the key role of oxidative stress in the pathogenesis of liver inflammation and metastasis. (HEPATOLOGY 2001;34:298-310.)

The Rh Factor: Past, Present, and Future

Liver metastasis causes nearly half of death from solid tumors. Metastatic lesions, to the liver in particular, can become detectable years or decades after primary tumor removal, leaving an uncertain long-term prognosis in patients. Prostate cancer (PCa), a prominent metastatic dormant cancer, has the worst prognosis when found in the liver compared to other metastatic sites. These metastatic nodules display a therapy resistance in the liver pro-metastatic microenvironment; the resistance appears to be conferred by both dormancy and independent of dormancy when the nodules emerge. Within the review, the molecular underpinnings of how the liver aids and protects PCa cells seeding, colonization and resistance will be discussed.