Host Stromal Cells Research Articles

Abstract LB-288: Restoration of peri-vascular nitric oxide gradient radio-sensitizes murine breast cancers via vascular normalization.

Abstract Background: Abnormal blood vessels in solid tumors hinder the delivery and efficacy of therapeutic agents. We and others have demonstrated that these physiological barriers can be overcome by normalization of the vascular (Science 307: 58, 2005). We have also reported that nitric oxide (NO) produced by endothelial NO synthase (eNOS) in vascular endothelial cells mediates angiogenesis and vessel maturation while ectopic NO production by tumor cells impairs tissue NO distribution and interferes with these processes (Nature Medicine 14:255, 2008). Inducible NO synthase (iNOS) is the most frequently expressed NOS in human tumors including breast cancers. Tumor hypoxia can also induce iNOS. Resulting heterogeneous NO distribution may contribute to the abnormality of tumor vasculature. Here, we determined the role of iNOS coming from tumor and/or stromal cells on the tumor vasculature and response to radiation therapy. Methods: Murine breast cancers (MCaIV and E0771) were grown orthotopically in the mammary fat pad in C3H and C57BL/6 mice, respectively. Perivascular cells were visualized using α-smooth muscle actin promoter driven green fluorescent protein or DsRed reporter mice. Tissue oxygenation was determined by a redox marker pimonizadole. To block iNOS in these tumors, we 1) silenced iNOS expression in tumor cells by shRNA, 2) used iNOS deficient mice to assess the role of iNOS in host stromal cells, and 3) used an iNOS selective inhibitor 1400W (10mg/kg/day). Fractionated radiation therapy (5 Gy/day × 3 days) was performed when mammary fat pad tumors reached 100 mm3. Results: Murine breast cancers express high levels of iNOS especially in vivo. The majority of iNOS expressing cells were tumor cells. Blood vessels have eNOS but not iNOS. Both genetic and pharmacological inhibition of iNOS resulted in an increased number of blood vessels (segments) and a decrease in the abnormal vessel dilation in the breast tumors. Furthermore, iNOS inhibition increased vessel maturation and suppressesd vascular hyperpermeability. Finally, iNOS silencing and inhibition decreased hypoxia and thus, improved tumor response to radiation therapy. In contrast, these parameters were not different between iNOS deficient and wild-type mice suggesting tumor cell-derived NO dominancy. Conclusion: In this study, we found that restoration of tissue distribution of NO in murine breast cancers to a normal pattern could normalize tumor vasculature both morphologically and functionally. The perivascular NO gradient significantly alleviated tissue hypoxia and improved efficacy of radiation therapy. Our data suggest that aberrant production of NO from tumor cells interfere with vessel maturation, and that restoration of tissue NO distribution by selective NOS modulation can improve tumor vascular function and facilitate concomitantly administered cytotoxic therapies. (Supported by NIH grant CA096915) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-288. doi:10.1158/1538-7445.AM2011-LB-288

Abstract 3114: Effects of tumor-stromal interactions on gene expression in panel of mouse tumor models

Abstract Tumor growth is not determined solely by the tumor cells but is governed by interactions between tumor cells and host stromal cells, including endothelial cell activation and fibroblastic stroma response. Tumor stroma profoundly influences many steps of tumor progression. In many human cancers, such as breast, prostate, and colon, the stroma comprises the majority of the tumor mass, as a hallmark of the clinical feature called desmoplasia. Numerous studies have showed that tumor-stromal cell interactions play crucial roles in supporting cancer progression and in promoting anticancer drug resistance by alternating gene expression profiles in both tumor and stromal cells through network tumor-stromal interactions in the tumor microenvironments. It has been challenging to obtain separate gene profiles for tumor and stroma with human tumor samples as both tumor and stromal cells share the same genome. In xenograft mouse tumor models, human tumor cells are supported by mouse host stromal microenvironment. Therefore, effects of tumor stromal integrations on gene expression can be profiled separately by taking advantage of this heterogeneous genetic makeup. To understand the underlying biological process of stroma in cancer and select relevant in vitro and in vivo model systems for various targeted anticancer drug discovery and development projects, we selected a panel of 30 commonly used xenograft tumor mouse models that are derived from human tumor cell lines of various cancer histotypes and conducted analyses of differential gene expression in both human cancer cells and mouse host stromal cells before and after their interactions in vivo by using quantitative PCR with mouse and human specific primers. Growing the human tumors as a continuous in vivo passage subcutaneously in immunodeficient mice permits stroma infiltration over a long time. We have examined a number of anticancer target genes involved in different signaling pathways, such as tumor angiogenesis, apoptosis and survival (Akt/mTOR signaling pathway). Our study results have demonstrated that tumor stromal interactions significantly regulate expression levels of various genes important in tumor progression and development of resistance to treatment in both tumor and stromal cells. Tumor animal models play a critical role in translating the bench science to the bedside medical care of cancer patients. Decisions for moving new anticancer agents into costly clinical investigations are mostly based on the preclinical results using xenograft mouse models. The results of this gene profiling approach could provide tools for studying tumor microenvironment and tumor stromal interactions in vivo to advance anticancer drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3114. doi:10.1158/1538-7445.AM2011-3114

Abstract 438: The CFP nude mouse as a host for color-coded imaging of the tumor microenvironment

Abstract We have developed a color-coded model to study the tumor microenvironment using cancer cells expressing RFP or GFP implanted in nude mice expressing cyan fluorescent protein (CFP). When RFP or GFP HCT-116 were injected subcutaneously in CFP expressing nude mice, CFP expressing cancer-associated fibroblasts migrated to the tumor site. CFP-expressing tumor blood vessels were observed in the tumor. Other CFP host stromal cells were observed throughout the tumor mass. When RFP HCT-116 cells were transplanted in the spleen of CFP nude mice, they formed tumors in the liver. When RFP HCT-116 cancer cells were transplanted in the tail vein of CFP nude mice and they formed tumors in the lung. CFP-expressing host cells were observed in these tumors. When the RFP mouse mammary cancer cell line (RFP MMT) was injected subcutaneously in the CFP nude mouse, numerous CFP-expressing fibroblasts and CFP-expressing blood vessels was observed in the resulting tumor. A large inflammatory population of different CFP-expressing white blood cells surrounded the necrotic area of the tumor suggesting a relationship of these cells to tumor necrosis. GFP and RFP HCT-116 cancer cells were co-injected subcutaneously in CFP-nude mice and tumor growth was followed for 21 days. This three-color model enabled visualization of the tumor mass and its neovasculature in addition to a detailed visualization of other host reactions. GFP HCT-116 cancer cells were initially injected subcutaneously in RFP-nude mice and tumors formed within 14 days. At this point, the tumor mass which consisted of GFP cancer cells and RFP stroma derived from the RFP nude mouse, was harvested and transplanted to a CFP-nude mouse. CFP host cells invaded the growing transplanted tumor containing dual-color cancer cells and RFP stroma. Thus the CFP-nude mouse adds another color to the pallet to study the human tumor microenvironment allowing 4 types of cells to be simultaneously imaged in tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 438. doi:10.1158/1538-7445.AM2011-438

Initial testing of lenalidomide by the pediatric preclinical testing program.

Lenalidomide, a novel immunomodulatory agent, is reported to modulate stem cell differentiation, and have direct antiproliferative activity as well as inhibit inflammation and hyperalgesia. On the basis of this varied pharmacological profile, lenalidomide is under investigation as a treatment for a range of oncologic indications. Lenalidomide was evaluated against the PPTP in vitro panel using 96-hr exposure at concentrations ranging from 1 nM to 10 µM. It was tested against the PPTP in vivo panels at a dose of 30 mg/kg administered orally (PO) once daily for a planned for 6 weeks. In vitro activity was not observed at concentrations up to 10 µM. Lenalidomide was well tolerated, and induced significant differences in EFS distribution compared to control in 7 of 37 (18.9%) of the evaluable solid tumor xenografts and in 0 of 8 (0%) of the evaluable ALL xenografts. The best response in the solid tumor panel was PD2 [progressive disease with growth delay (EFS T/C > 1.5)], observed in 4 of 37 (10.8%) solid tumor xenografts. A single ALL xenograft showed a PD2 response. Direct antiproliferative effects of lenalidomide were not observed in vitro. In vivo lenalidomide demonstrated low activity against tumors in immune-deficient mice. Our results suggest that lenalidomide's utility in the pediatric clinical setting may depend upon its ability to induce antitumor activity through effects on host immune and stromal cells rather than through direct effects on tumor cells.

Tumor-secreted PGE2 Inhibits CCL5 Production in Activated Macrophages through cAMP/PKA Signaling Pathway

One of the major characteristics of tumors is their ability to evade immunosurveillance through altering the properties and functions of host stromal and/or immune cells. CCL5 has been shown to play important roles in T cell proliferation, IFN-γ, and IL-2 production, which promotes the differentiation and proliferation of Th1 cells important for immune defense against intracellular infection. In this study we found that tumor-bearing mice were more susceptible to bacterial infection and showed reduced CCL5 levels in serum during endotoxic shock. Our data further demonstrated that the soluble factors secreted by mammary gland tumor cells but not normal mammary gland epithelial cells inhibited CCL5 expression in macrophages in response to LPS, but not to TNF-α stimulation. The inhibitory effect of tumor-secreted molecules on LPS-induced CCL5 expression was regulated at the post-transcriptional level. Blocking PGE(2) synthesis by NS398 or through the use of PGE(2) receptor antagonists AH-6809 (EP2 antagonist) and AH-23848 (EP4 antagonist) completely reversed the inhibitory effect of tumor-conditioned medium (TCM) on LPS-induced CCL5 expression. Moreover, PGE(2) and the cAMP analog forskolin could mimic tumor-mediated CCL5 inhibition, and the inhibitory effects of TCM, PGE(2), and cAMP analog on LPS-induced CCL5 expression could be completely reversed by the PKA inhibitor H89. Furthermore, blocking PGE(2) synthesis in vivo led to partial recovery of CCL5 production during endotoxic shock. Taken together, our data indicate that PGE(2) secreted from breast cancer cells suppresses CCL5 secretion in LPS-activated macrophages through a cAMP/PKA signaling pathway, which may result in suppression of host immune responses against subsequent bacterial infection.

Kinetics of host cell recruitment during dissemination of diffuse malignant peritoneal mesothelioma.

Diffuse malignant mesothelioma is an aggressive tumor which displays a median survival of 11.2 months and a 5-year survival of less than 5% emphasizing the need for more effective treatments. This study uses an orthotopic model of malignant mesothelioma established in syngeneic, immunocompetent C57Bl/6 mice which produce malignant ascites and solid tumors that accurately replicate the histopathology of the human disease. Host stromal and immune cell accumulation within malignant ascites and solid tumors was determined using immunofluorescent labeling with confocal microscopy and fluorescence-activated cell sorting. An expression profile of cytokines and chemokines was produced using quantitative real-time PCR arrays. Tumor spheroids and solid tumors show progressive growth and infiltration with host stromal and immune cells including macrophages, endothelial cells, CD4+ and CD8+ lymphocytes, and a novel cell type, myeloid derived suppressor cells (MDSCs). The kinetics of host cell accumulation and inflammatory mediator expression within the tumor ascites divides tumor progression into two distinct phases. The first phase is characterized by progressive macrophage and T lymphocyte recruitment, with a cytokine profile consistent with regulatory T lymphocytes differentiation and suppression of T cell function. The second phase is characterized by decreased expression of macrophage chemotactic and T-cell regulating factors, an increase in MDSCs, and increased expression of several cytokines which stimulate differentiation of MDSCs. This cellular and expression profile suggests a mechanism by which host immune cells promote diffuse malignant mesothelioma progression.Electronic supplementary materialThe online version of this article (doi:10.1007/s12307-010-0048-1) contains supplementary material, which is available to authorized users.

Abstract 3847: Metastasizing host stromal cells potentiate colonization of metastatic tumors

Abstract Metastatic cancer cells only grow in the secondary sites with a permissive microenvironment. Here, we show that the metastatic cells can also carry their own “soil”, including activated fibroblasts from the primary site. Moreover, we found that these non-malignant carryover host-derived cells can potentiate metastatic cell colonization in the lungs and increase cancer's metastatic efficiency. Materials and Methods: Using the isolated renal perfusion model we were able to collect fragments shed by the primary tumor and assess their viability. Next, we adjusted the parabiosis model to transplant a 2 cm Actb-GFP+ skin graft onto a wild type mouse. Tumors were allowed to grow in the graft and metastasize to the lung. GFP+ cells carried over by the tumor to the lung in the lung were characterized using immunohistochemistry. In a spontaneous metastases model we used two lung tumor cell lines (LLC1 and LA-P0297) in combination with human carcinoma associated fibroblasts to study the modulation of the metastatic efficiency by stromal cells. Results: We show here that, in addition to single cells (81%), tumors shed fragments consisting of both tumor and host stromal cells. These heterogeneous fragments are more viable in the circulation compared to tumor cells alone (p < 0.05) and their stromal component proliferates in the metastic site for up to seven days. Next, we assessed the nature of these host stromal cells using immunohistochemistry and indentified 80% of them being of fibroblast origin. When we selectively depleted the host stromal cells in the tumor microenvironment the spontaneous formation of metastases was significantly reduced three weeks after the primary tumor resection as compared to control mice (p < 0.05). Also, in mice with metastases, survival was significantly decreased after co-implantation of CAFs with LLC1 compared to mice implanted with cancer cells alone. This effect could be reversed by selective depletion of the “circulating” CAFs after primary tumor removal (p < 0.01). Discussion: We found that tumor-associated stromal cells can escape primary tumors along with cancer cells, survive in blood circulation and in metastatic nodules. Pre-existence of a tissue-like structure in the form of heterotypic cell fragments may increase the viability of cancer cells in blood circulation and promote metastasis efficiency at the secondary site. How this mechanism contributes to the organo-tropism of certain metastatic tumors is not known. But demonstration of the involvement of some of the same tumor “soil” in both local and distant tumor invasion has important conceptual and clinical implications for colonization during metastasis progression. 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 3847.

Abstract 600: Targeting the hedgehog pathway by LNA (locked nucleic acid) oligonucleotide-based GLI2 RNA antagonists, EZN-4482 and EZN-4496, in vitro and in vivo

Abstract Background: Aberrent activation of hedgehog (Hh) signaling has been implicated in the initiation and growth of a number of human malignancies, including those derived from skin, lung, liver, prostate and breast. The GLI family proteins are zinc finger transcription factors and the downstream effectors of Hh pathway, controling expression of the target genes such as GLI1, Ptch1, Cyclin D, and BCL2. Since amplification, overexpression of Hh ligand, and mutations of components along the Hh pathway are found in cancer and cancer stem cells, specific down regulation of GLIs with antisense molecules may offer a novel and effective therapeutic approach for cancer treatment. We report here the biological activities of LNA-based GLI2 mRNA antagonist, EZN-4482 and EZN-4496. Methods: In vitro, the ability of the antagonists to down modulate their direct target, GLI2 mRNA, and to inhibit cell growth was evaluated by qRT-PCR and MTS, respectively, in multiple cell lines of various origins. To identify and investigate the molecular events associated with targeted downmodulation of GLI2, expression of Hh pathway components and downstream effectors were analyzed by gene expression profiling and immnomoblotting analysis. In vivo, GLI2 mRNA down-modulation in PC3 xenograft tumor was evaluated after intravenous administration of the antagonists. The effect of antagonists on tumor growth and survival of mice were evaluated in PC3 prostate tumor grown on the flank and in A549 lung carcinoma cells that had metastasized to the liver respectively. Results: Both antagonists are specific and potent down-modulators of GLI2 mRNA (by transfection, IC50 < 2 nM). Down-modulation of GLI2 mRNA with or without transfection was shown to result in reduction of GLI2 protein in a human breast cancer cell line, HCC-1937, and human prostate cancer cell lines, 15PC3 and C4-2b. In mice bearing PC3 xenograft tumors, systemic administration of EZN-4482 resulted in down-modulation of GLI2, GLI1, and PTCH1 in both PC3 and host stromal cells. Additionally, we show that EZN-4482 prolonged the survival of animals with A549 cell derived liver metastasis derived from A549 cells. Conclusions: EZN-4482 and EZN-4496 are potent and specific GLI2 antagonists in vitro and in vivo. The possible mechanisms of efficacy in vivo may include inhibition of mRNA levels of GLI1/2 and PTCH1 in mouse stromal cells. Therefore, targeted down-modulation of GLI2 using the LNA-based mRNA antagonists may be an effective therapy to treat a broad spectrum of cancers including ones that fail treatment with SMO inhibitor therapy. 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 600.

Abstract 5218: γ-secretase inhibitor PF-03084014 impairs Notch signaling and induces antiangiogenic and antitumor effects in breast cancer models

Abstract PF-03084014, a small molecule γ-secretase inhibitor (GSI), has previously been shown to inhibit Notch signaling and demonstrated antitumor efficacy. The aim of this report is to gain insights into the mechanism PF-03084014-induced activity. The cell-based evaluation of PF-03084014 across 30 breast cancer (BC) cell lines indicated significant growth inhibition in only a subset of cell lines (3/30). In contrast, PF-03084014 demonstrated cell cycle arrest and induction of apoptosis by FACS analysis across the majority of a panel of T-acute lymphoblastic leukemia (T-ALL) cell lines. In vivo BrdU uptake of tumor and [18F]FLT-PET imaging studies demonstrated that PF-03084014 at an efficacious dose (120 mg/kg) caused cell cycle arrest in Sup T1 tumors but not in MDA-MB-231 xenografts, although PF-03084014 suppressed the expression of Notch target genes and tumor growth in both models. These results suggest that the primary mechanism(s) for PF-03084014-induced efficacy in T-ALL and BC models may be different, possibly because the tumor-host microenvironment in the BC model plays an important role during Notch signaling activation. IHC analysis in a panel of solid tumors demonstrated that Notch ligands including jagged 1, 2 and Dll4 were predominately expressed in the host vasculature and stromal cells. To investigate the potential antiangiogenic activity of PF-03084014, an in vitro endothelial cell/fibroblast co-culture tube formation assay was utilized. PF-03084014 disrupted the multicellular lumen-like structures at 100 nM, indicating defective differentiation of endothelial cells in early stage angiogenesis. Ultrasound imaging was utilized to further investigate the antiangiogenic properties of PF-03084014 including both the quantitative measurement of the total tumor vessel volume using 3-D scanning methods and an assessment tumor blood vessel function by measuring blood flow after iv injection of microbubble contrast agents. When MDA-MB-231 tumor bearing mice were administered with an efficacious dose of PF-03084014 consecutively for 4 days, ultrasound imaging revealed a significant (p< 0.05) decrease of total tumor vessel volume and function compared to the vehicle treatment. A subsequent FITC-lectin vascular perfusion assay was performed to confirm the reduction in functional microvasculature by PF-03084014. In addition to the antiangiogenic effect, we also observed treatment-induced caspase 3 activation and Ki67 suppression in the MDA-MB-231 tumor model (2 days after treatment), suggest the GSI also induces apoptosis and cytoreductive activity. Collectively, our data provide insights that activation of Notch signaling requires the interaction of tumor cells with their host environment in solid tumors, and impairing Notch signaling by PF-03084014 results in significant antitumor efficacy in the breast cancer xenografts. 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 5218.

Abstract 705: Crosstalk with stromal fibroblasts contributes to EGFR inhibitor resistance in colorectal cancer cells

Abstract The mechanism for resistance to EGFR targeted therapies in colorectal cancer (CRC) with intact KRAS is still unknown. Host stromal cells contribute to tumorigenicity; however their effect on the sensitivity to EGFR targeted therapies has still to be determined. We hypothesized that crosstalk between stromal fibroblasts and CRC cancer cells contributes to cetuximab resistance via activation of the HGF/MET axis. Primary CRC associated fibroblasts (CAFs) were isolated and expanded from five surgical specimens. Fibroblasts were enriched by magnetic activated cell sorting (MACS®) and activated fibroblast lineage was confirmed by alpha smooth muscle actin (SMA) immunofluorescence. Supernatant HGF content and cellular mRNA expression levels in primary CRC CAFs, an embryonic lung fibroblast cell line (MRC-5), and a normal colon fibroblast cell line (CCD18) were determined by ELISA and quantitative RT-PCR. The effect of fibroblasts or HGF (75ng/ml) on the CRC cell lines LIM1215 and DiFi treated with cetuximab (5nM) for 72 hours was determined by co-culture using Transwells and WST-1 cell proliferation assays. Alterations in proliferation were compared by ANOVA. Mean fibroblast HGF secretion (8.3 ng/ml) and relative mRNA expression (2.0) could be augmented by co-culture with conditioned media (CM) from the CRC cell LIM1215 (14.1 ng/ml and 4.1, respectively) (Table). Further, co-culture of CRC cells with HGF, CCD18 cells, or primary CAFs reversed cetuximab-induced growth inhibition. LIM1215 proliferation was inhibited by cetuximab (55.3 ± 14.4%) which could be reversed by co-culture with either HGF (93.1 ± 10.2%) or CCD18 cells (107.2 ± 10.8%) (p<0.01 for both). Stromal-tumor interactions may play an important role in CRC resistance to cetuximab via the HGF/MET axis. Inhibition of both EGFR and MET may be necessary to realize the full potential of EGFR targeted therapies in CRC.Supernatant HGF levels and relative mRNA expression in fibroblasts treated with LIM1215 CM LIM1215 (Control)MRC5CCD18CAF3CAF4CAF5CAF6HGF (ng/ml) Ctrl<0.11.217.310.97.24.08.9LIM1215-CM<0.10.840.015.08.45.015.5 HGF mRNA Ctrl00.81.82.63.01.6 LIM1215-CM01.07.65.44.91.8 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 705.

Tumor-secreted PGE2 inhibits CCL5 production from activated macrophages through cAMP signaling pathway (100.6)

Abstract It is well known that tumor cells could evade immuosurveillance by dampening host immune responses and shaping host stromal cells to favor its growth. CCL5 has been shown to play important roles in T cell proliferation, IFN-g and IL-2 production, which promotes differentiation and proliferation of Th1 cells important for immune defense against intracellular infection. Here we report that the CCL5 mRNA expression and protein production were significantly reduced in tumor-bearing mice compared with tumor-free mice during endotoxic shock. Our data further demonstrate that soluble factors secreted from tumor cells inhibited CCL5 mRNA expression and protein production in LPS-stimulated macrophages. The inhibition of CCL5 expression was regulated at the level of post-transcription. NS398, a selective inhibitor of enzymatic activity of cyclooxygenase 2 in PGE2 synthesis, could reverse the inhibitory effect of tumor cells on LPS-induced CCL5 expression. Moreover, PGE2 and cAMP analog Forskolin could mimic the inhibitory effect of tumor cells on CCL5 expression and blockade of cAMP signaling by H89 completely reversed the inhibitory effect. Taken together, our data indicate that PGE2 secreted from tumor cells suppresses CCL5 expression in LPS-activated macrophages through a cAMP signaling pathway, which may result in suppression of host immune responses against subsequently bacterial infection.

Angiotensin II type 2 receptor signaling significantly attenuates growth of murine pancreatic carcinoma grafts in syngeneic mice

BackgroundPancreatic cancer is one of the most aggressive human malignancies, with a very poor prognosis. To evaluate the effect of angiotensin II (Ang II) type 2 receptor (AT2) expression in the host's body on the growth of pancreatic carcinoma, we have investigated the growth of mouse pancreatic ductal carcinoma grafts in syngeneic wild type and AT2 receptor-deficient (AT2-KO) mice.MethodsThe role of AT2 receptor-signaling in stromal cells on the growth of murine pancreatic carcinoma cells (PAN02) was studied using various in vitro and in vivo assays. In vivo cell proliferation, apoptosis, and vasculature in tumors were monitored by Ki-67 immunostaining, TUNEL assay, and von Willebrand factor immunostaining, respectively. In the co-culture study, cell proliferation was measured by MTT cell viability assay. All the data were analyzed using t-test and data were treated as significant when p < 0.05.ResultsOur results show that the growth of subcutaneously transplanted syngeneic xenografts of PAN02 cells, mouse pancreatic ductal carcinoma cells derived from the C57/BL6 strain, was significantly faster in AT2-KO mice compared to control wild type mice. Immunohistochemical analysis of tumor tissue revealed significantly more Ki-67 positive cells in xenografts grown in AT2-KO mice than in wild type mice. The index of apoptosis is slightly higher in wild type mice than in AT2-KO mice as evaluated by TUNEL assay. Tumor vasculature number was significantly higher in AT2-KO mice than in wild type mice. In vitro co-culture studies revealed that the growth of PAN02 cells was significantly decreased when grown with AT2 receptor gene transfected wild type and AT2-KO mouse-derived fibroblasts. Faster tumor growth in AT2-KO mice may be associated with higher VEGF production in stromal cells.ConclusionsThese results suggest that Ang II regulates the growth of pancreatic carcinoma cells through modulating functions of host stromal cells; Moreover, Ang II AT2 receptor signaling is a negative regulator in the growth of pancreatic carcinoma cells. These findings indicate that the AT2 receptor in stromal fibroblasts is a potentially important target for chemotherapy for pancreatic cancer.

Of mice and (wo)men: Mouse models of breast cancer metastasis to bone

Of mice and (wo)men: Mouse models of breast cancer metastasis to bone

Stromal cell‐derived CSF‐1 blockade prolongs xenograft survival of CSF‐1‐negative neuroblastoma

The molecular mechanisms of tumor-host interactions that render neuroblastoma (NB) cells highly invasive are unclear. Cancer cells upregulate host stromal cell colony-stimulating factor-1 (CSF-1) production to recruit tumor-associated macrophages (TAMs) and accelerate tumor growth by affecting extracellular matrix remodeling and angiogenesis. By coculturing NB with stromal cells in vitro, we showed the importance of host CSF-1 expression for macrophage recruitment to NB cells. To examine this interaction in NB in vivo, mice bearing human CSF-1-expressing SK-N-AS and CSF-1-negative SK-N-DZ NB xenografts were treated with intratumoral injections of small interfering RNAs directed against mouse CSF-1. Significant suppression of both SK-N-AS and SK-N-DZ NB growth by these treatments was associated with decreased TAM infiltration, matrix metalloprotease (MMP)-12 levels and angiogenesis compared to controls, while expression of tissue inhibitors of MMPs increased following mouse CSF-1 blockade. Furthermore, Tie-2-positive and -negative TAMs recruited by host CSF-1 were identified in NB tumor tissue by confocal microscopy and flow cytometry. However, host-CSF-1 blockade prolonged survival only in CSF-1-negative SK-N-DZ NB. These studies demonstrated that increased CSF-1 production by host cells enhances TAM recruitment and NB growth and that the CSF-1 phenotype of NB tumor cells adversely affects survival.

The Role of Stromal Cells in Tumor Metastases.

Abstract Background: In 1889, Stephen Paget hypothesized that metastatic cells - the “seeds” - only grow in the secondary sites - the “soil” - with a permissive microenvironment. Here, we show that the “seeds” can carry their own “soil” - activated fibroblasts from primary site of metastatic tumors. Moreover, we report that carryover of these non-malignant host-derived cells during metastatic cell colonization in the lungs can increase cancer's metastatic efficiency.Materials and Methods: We use multiple animal models and two metastatic tumor cell lines (LLC1 and LA-P0297) to study the composition, origin and role of fragments shed by tumors in the metastatic process.Results: we have shown that, in addition to single cells (81%), tumors shed fragments consisting of both tumor and host cells. We show that these fragments are more viable in the circulation compared to tumor cells alone (p&amp;lt;0.05). When we selectively deplete the host cells in the tumor microenvironment the spontaneous formation of metastases is significantly reduced three weeks after primary tumor resection compared to control mice (p&amp;lt;0.05). Next, survival was significantly increased in mice with metastases after co-implantation of carcinoma-associated fibroblasts (CAFs) isolated from breast cancer patients together with LLC1 cells, but had no effect in mice implanted with LLC1 cells alone (p&amp;lt;0.01) Lastly, we have assessed the nature of these host stromal cells using immunohistochemistry and RT-PCR and indentified the majority of them as being of mesenchymal/fibroblast lineage.Discussion: We found that tumor-associated stromal cells can escape primary tumors along with cancer cells, survive in blood circulation and in metastatic nodules. Pre-existence of a tissue-like structure in the form of heterotypic cell fragments may increase the viability of cancer cells in blood circulation and modulate metastasis efficiency. How this mechanism contributes to the organo-tropism of certain metastatic tumors is not known. But demonstration of the involvement of some of the same tumor “soil” in both local and distant tumor invasion has important conceptual and clinical implications for colonization during metastasis progression. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6151.

Abstract C57: The role of stromal cells in tumor metastases

Abstract Background: In 1889, Stephen Paget hypothesized that metastatic cells - the “seeds” - only grow in the secondary sites - the soil” - with a permissive microenvironment. Here, we show that the “seeds” can carry their own “soil” - activated fibroblasts from primary site of metastatic tumors. Moreover, we report that carryover of these non-malignant host-derived cells during metastatic cell colonization in the lungs can increase cancer's metastatic efficiency. Materials and Methods: We use multiple animal models and two lung tumor cell lines (LLC1 and LA-P0297) to study the composition, origin and role of fragments shed by tumors in the metastatic process. Results: We have shown that, in addition to single cells (81%), tumors shed fragments consisting of both tumor and host cells. We show that these fragments are more viable in the circulation compared to tumor cells alone (p&amp;lt;0.05).When we selectively deplete the host cells in the tumor microenvironment the spontaneous formation of metastases is significantly reduced three weeks after primary tumor resection compared to control mice (p&amp;lt;0.05). Next, survival was significantly increased when we deplete host-cells in mice with metastases after co-implantation of CAFs with LLC1, but had no effect in mice implanted with cancer cells alone (p&amp;lt;0.01) Lastly, we have assessed the nature of these host stromal cells using immunohistochemistry and RT-PCR and identified them being of fibroblast origin. Discussion: We found that tumor-associated stromal cells can escape primary tumors along with cancer cells, survive in blood circulation and in metastatic nodules. Pre-existence of a tissue-like structure in the form of heterotypic cell fragments may increase the viability of cancer cells in blood circulation and modulate metastasic efficiency. How this mechanism contributes to the organo-tropism of certain metastatic tumors is not known, but demonstration of the involvement of tumor “soil” in both the primary tumor as well as the metastatic site, has important conceptual and clinical implications for colonization during metastasis progression. Citation Information: Cancer Res 2009;69(23 Suppl):C57.

Crosstalk to Stromal Fibroblasts Induces Resistance of Lung Cancer to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors

Lung cancers with epidermal growth factor receptor (EGFR)-activating mutations show good clinical response to gefitinib and erlotinib, selective tyrosine kinase inhibitors (TKI) to EGFR, but these tumors invariably develop drug resistance. Host stromal cells have been found to have a considerable effect on the behavior of cancer cells. Little is known, however, about the role of host cells on the sensitivity of cancer cells to receptor TKIs. We have therefore assessed the effect of crosstalk between stromal cells and lung cancer cells harboring EGFR mutations on susceptibility to EGFR-TKIs. We evaluated the gefitinib sensitivity of lung cancer cells with EGFR-activating mutations, PC-9 and HCC827, when cocultured with fibroblasts and coinjected into severe combined immunodeficient mice. We also examined the effect of lung cancer cells to fibroblast recruitment. Both human fibroblast cell lines and primary cultured fibroblasts produced various levels of hepatocyte growth factor (HGF). Lung cancer cells markedly recruited fibroblasts. The lung cancer cells became resistant to EGFR-TKIs when cocultured in vitro with HGF-producing fibroblasts and coinjected into severe combined immunodeficient mice. Importantly, combined use of gefitinib plus anti-HGF antibody or the HGF antagonist, NK4, successfully overcame the fibroblast-induced EGFR-TKI resistance both in vitro and in vivo. Colocalization of fibroblasts and HGF was detected in both xenograft tumors in mouse model and lung cancer patient specimens. These findings indicate that crosstalk to stromal fibroblasts plays a critical role in lung cancer resistance to EGFR-TKIs and may be an ideal therapeutic target in lung cancer with EGFR-activating mutations.

Endometrial cancer invasion depends on cancer‐derived tumor necrosis factor‐α and stromal derived hepatocyte growth factor

Cancer invasion is an outcome of interactions of the cancer and the host cell. It is now becoming increasingly clear that ovarian hormones have a huge influence on such intercommunications in various types of cancers. Estrogen is known to aggravate the aggressiveness of the endometrial cancer whereas progesterone seems to act as a negative factor. Insight into the mode of ovarian hormonal actions could come from the studies of its regulation of the paracrine interactions between the endometrial cancer and the normal stromal cells during the cancer invasion. In this context, we report here that estrogen promotes the endometrial cancer invasion by inducing humoral interactions between the cancer and the stromal cells, i.e., estrogen stimulates tumor necrosis factor-alpha expression from the endometrial cancer cells, which, in turn, induces the stromal expression of hepatocyte growth factor (HGF), conferring the enhanced NK4 (HGF-antagonist/angiogenesis inhibitor)-sensitive invasion characteristic of the endometrial cancer cells. Additionally, we demonstrate a close correlation of the invasion of endometrial cancer cells with the expression and dimerization of integrin alpha(v)beta(5) as well as the activation of focal adhesion kinase as the consequences of paracrine interactions. Thus, understanding of paracrine interactions of cancer cells with host stromal cells can yield new insight into the architecture and function of cancer invasion and metastasis, leading to a development of a new cancer therapeutic intervention.

Vascular determinants of cancer stem cell dormancy—do age and coagulation system play a role?

The inability of tumour-initiating cancer stem cells (CSCs) to bring about a net increase in tumour mass could be described as a source of tumour dormancy. While CSCs may be intrinsically capable of driving malignant growth, to do so they require compatible surroundings of supportive cells, growth factors, adhesion molecules and energy sources (e.g. glucose and oxygen), all of which constitute what may be referred to as a 'permissive' CSC niche. However, in some circumstances, the configuration of these factors could be incompatible with CSC growth (a 'non-permissive' niche) and lead to their death or dormancy. CSCs and their niches may also differ between adult and paediatric cancers. In this regard the various facets of the tumour-vascular interface could serve as elements of the CSC niche. Indeed, transformed cells with an increased tumour-initiating capability may preferentially reside in specific zones adjacent to tumour blood vessels, or alternatively originate from poorly perfused and hypoxic areas, to which they have adapted. CSCs themselves may produce increased amounts of angiogenic factors, or rely for this on their progeny or activated host stromal cells. It is likely that 'vascular' properties of tumour-initiating cells and those of their niches may diversify and evolve with tumour progression. The emerging themes in this area include the role of vascular (and bone marrow) aging, vascular and metabolic comorbidities (e.g. atherosclerosis) and the effects of the coagulation system (both at the local and systemic levels), all of which could impact the functionality of CSCs and their niches and affect tumour growth, dormancy and formation of occult as well as overt metastases. In this article we will discuss some of the vascular properties of CSCs relevant to tumour dormancy and progression, including: (i) the role of CSCs in regulating tumour vascular supply, i.e the onset and maintenance of tumour angiogenesis; (ii) the consequences of changing vascular demand (vascular dependence) of CSC and their progeny; (iii) the interplay between CSCs and the vascular system during the process of metastasis, and especially (iv) the impact of the coagulation system on the properties of CSC and their niches. We will use the oncogene-driven expression of tissue factor (TF) in cancer cells as a paradigm in this regard, as TF represents a common denominator of several vascular processes that commonly occur in cancer, most notably coagulation and angiogenesis. In so doing we will explore the therapeutic implications of targeting TF and the coagulation system to modulate the dynamics of tumour growth and tumour dormancy.

Tumor stroma marker endosialin (Tem1) is a binding partner of metastasis‐related protein Mac‐2 BP/90K

Tumor development involves complex bidirectional interactions between tumor cells and host stromal cells. Endosialin (Tem1) has been identified as a highly O-glycosylated transmembrane glycoprotein, which is specifically expressed by tumor vessel-associated pericytes and stromal fibroblasts of a wide range of human tumors. Recent experiments in endosialin-deficient mice have unraveled a critical role of endosialin in site-specific tumor progression and metastasis. To molecularly understand the mechanisms of endosialin function, we aimed to identify extracellular endosialin ligands and identified Mac-2 BP/90K as a specific interaction partner. Detailed biochemical analyses identified a C-terminal fragment of Mac-2 BP/90K, which was shown to contain binding sites for galectin-3, and collagens as the structures responsible for endosialin binding. Subsequent expression analysis of Mac-2 BP/90K in vivo revealed weak or no expression in most normal tissues and strong up-regulation in tumor cells of human neoplastic tissues. Intriguingly, the expression patterns of Mac-2 BP/90K and endosialin were mutually exclusive in all human tissues. Correspondingly, loss-of-function adhesion experiments of Mac-2 BP/90K-expressing tumor cells on endosialin-expressing fibroblasts revealed a repulsive outcome of the Mac-2 BP/90K interaction. Taken together, the experiments identify a novel repulsive interaction between endosialin on stromal fibroblasts and Mac-2 BP/90K on tumor cells.