Later Stages Of Tumor Growth Research Articles

Infiltration of innate and adoptive lymphoid cells in 4T1 and MC4-L2 breast cancer models.

Innate lymphoid cells (ILCs) are tissue-resident lymphocytes that have vital roles in activating further immune responses. However, due to their tumor-induced diversity, we decided to examine ILCs, T cells, and the associated cytokines in mouse models of breast cancer. 4T1 and MC4-L2 cells were used to induce triple-negative and hormone-receptor-positive breast cancer, respectively. Tumor tissue was resected at early and late stages of tumor growth and used for further analysis. Total RNA was extracted and used in Real-Time PCR to analyze the expression of IFN-γ, IL-4, IL-10, IL-13, and IL-22. Tumor tissue was digested and used in a flow cytometric assay. H&E staining was used to examine the pathology of tumor progression. Both tumor models showed a notable increase in T-cell frequency at the early stage of tumor growth. However, as the tumors progressed, the frequency of T cells significantly decreased, while the ILC component exhibited a significant increase in tumor progression. Gene analysis indicated a significant increase in the inflammatory to anti-inflammatory cytokine ratio during tumor progression in the tumor model. In contrast, this ratio was considerably reduced in advanced MC4-L2 tumors. Both tumor models showed the development of invasive breast carcinoma and lung metastasis in advanced tumors. Our study highlighted the expansion of ILCs during tumor progression in two distinct breast cancer models with different immunogenicity. These findings suggest that ILCs may actively modulate the tumor microenvironment during the advanced stage of tumor growth.

Tumor endothelial cell-derived Sfrp1 supports the maintenance of cancer stem cells via Wnt signaling

Cancer stem cells (CSCs), which are critical targets for cancer therapy as they are involved in drug resistance to anticancer drugs, and metastasis, are maintained by angiocrine factors produced by particular niches that form within tumor tissue. Secreted frizzled-related protein 1 (Sfrp1) is an extracellular protein that modulates Wnt signaling. However, the cells that produce Sfrp1 in the tumor environment and its function remain unclear. We aimed to elucidate angiocrine factors related to CSC maintenance, focusing on Sfrp1. Although Sfrp1 is a Wnt pathway-related factor, its impact on tumor tissues remains unknown. We investigated the localization of Sfrp1 in tumors and found that it is expressed in some tumor vessels. Analysis of mice lacking Sfrp1 showed that tumor growth was suppressed in Sfrp1-deficient tumor tissues. Flow cytometry analysis indicated that CSCs were maintained in the early tumor growth phase in the Sfrp1 knockout (KO) mouse model of tumor-bearing cancer. However, tumor growth was inhibited in the late tumor growth phase because of the inability to maintain CSCs. Real-time PCR results from tumors of Sfrp1 KO mice showed that the expression of Wnt signaling target genes significantly decreased in the late stage of tumor growth. This suggests that Sfrp1, an angiocrine factor produced by the tumor vascular niche, is involved in Wnt signaling-mediated mechanisms in tumor tissues.

Murine cytotoxic CD4+ T cells in the tumor microenvironment are at a hyper-maturation stage of Th1 CD4+ T cells sustained by IL-12.

The roles of tumor-infiltrating CD4+Foxp3- T cells are not well characterized due to their plasticity of differentiation, and varying levels of activation or exhaustion. To further clarify this issue, we used a model featuring subcutaneous murine colon cancer and analyzed the dynamic changes of phenotype and function of the tumor-associated CD4+ T-cell response. We found that, even at a late stage of tumor growth, the tumor-infiltrating CD4+Foxp3- T cells still expressed effector molecules, inflammatory cytokines and molecules that are expressed at reduced levels in exhausted cells. We used microarrays to examine the gene-expression profiles of different subsets of CD4+ T cells and revealed that the tumor-infiltrating CD4+Foxp3- T cells expressed not only type 1 helper (Th1) cytokines, but also cytolytic granules such as those encoded by Gzmb and Prf1. In contrast to CD4+ regulatory T cells, these cells exclusively co-expressed natural killer receptor markers and cytolytic molecules as shown by flow-cytometry studies. We used an ex vivo killing assay and proved that they could directly suppress CT26 tumor cells through granzyme B and perforin. Finally, we used pathway analysis and ex vivo stimulation to confirm that the CD4+Foxp3- T cells expressed higher levels of IL12rb1 genes and were activated by the IL-12/IL-27 pathway. In conclusion, this work finds that, in late-stage tumors, the tumor-infiltrating lymphocyte population of CD4+ cells harbored a sustained, hyper-maturated Th1 status with cytotoxic function supported by IL-12.

Delineating the twin role of autophagy in lung cancer.

Autophagy represents an intracellular defense mechanism equipped within each eukaryotic cells to enable them to cope with variety of physical, chemical, and biological stresses. This mechanism helps to restore the homeostasis and preserve the cellular integrity and function of the cells. In these conditions, such as hypoxia, nutrient deprivation, inhibition of protein synthesis or microbial attack, the process of autophagy is upregulated to maintain cellular homeostasis. The role of autophagy in cancer is an intriguing topic which needs further exploration. This process of autophagy has been many times referred as a double-edged sword in the process of tumorigenesis. In the initial stages, it may act as a tumor suppressor and enable to quench the damaged organelles and harmful molecules generated. In more advanced stages, autophagy has been shown to act as a tumor-promoting system as it may help the cancer cells to cope better with stressful microenvironments. Besides this, autophagy has been associated with development of resistance to anticancer drugs as well as promoting the immune evasion in cancer cells, representing a serious obstacle in cancer treatment and its outcome. Also, autophagy is associated with hallmarks of cancer that may lead to activation of invasion and metastasis. The information on this twin role needs further exploration and deeper understanding of the pathways involved. In this review, we discuss the various aspects of autophagy during tumor development, from early to late stages of tumor growth. Both the protective role of autophagy in preventing tumor growth and the underlying mechanisms adopted with evidence from past studies have been detailed. Further, the role of autophagy in conferring resistance to distinct lung cancer treatment and immune shielding properties has also been discussed. This is essential for further improving on treatment outcome and success rates.

A TLR4 agonist improves immune checkpoint blockade treatment by increasing the ratio of effector to regulatory cells within the tumor microenvironment

Brucella lumazine synthase (BLS) is a homodecameric protein that activates dendritic cells via toll like receptor 4, inducing the secretion of pro-inflammatory cytokines and chemokines. We have previously shown that BLS has a therapeutic effect in B16 melanoma-bearing mice only when administered at early stages of tumor growth. In this work, we study the mechanisms underlying the therapeutic effect of BLS, by analyzing the tumor microenvironment. Administration of BLS at early stages of tumor growth induces high levels of serum IFN-γ, as well as an increment of hematopoietic immune cells within the tumor. Moreover, BLS-treatment increases the ratio of effector to regulatory cells. However, all treated mice eventually succumb to the tumors. Therefore, we combined BLS administration with anti-PD-1 treatment. Combined treatment increases the outcome of both monotherapies. In conclusion, we show that the absence of the therapeutic effect at late stages of tumor growth correlates with low levels of serum IFN-γ and lower infiltration of immune cells in the tumor, both of which are essential to delay tumor growth. Furthermore, the combined treatment of BLS and PD-1 blockade shows that BLS could be exploited as an essential immunomodulator in combination therapy with an immune checkpoint blockade to treat skin cancer.

The multifaceted roles of nucleophagy in cancer development and therapy.

Autophagy is an evolutionarily conserved process in which the cell degrades its own components and recycles the biomolecules for survival and homeostasis. It is an important cellular process to eliminate pathogens or damaged organelles. Nucleophagy, also termed as nuclear autophagy, is a more recently described subtype of autophagy, in which nuclear components, such as nuclear lamina and DNA, are to be degraded. Nucleophagy plays a double-facet role in the development of cancer. On one hand, the clearance of damaged DNA or nuclear structures via autophagic pathway is crucial to maintain nuclear integrity and prevent tumorigenesis. On the other hand, in later stages of tumor growth, nucleophagy may facilitate cancer cell survival and metastasis in the nutrient-depleted microenvironment. In this review, we discuss the relationship between nucleophagy and cancer along with potential intervention methods to target cancer through manipulating nucleophagy. Given the known observations about nucleophagy, it could be promising to target different nuclear components during the processes of nucleophagy, especially nuclear lamina. Further research on investigating the role of nucleophagy in oncological context could focus on dissecting its remaining molecular pathways and their connection to known tumor suppressors.

Study of photobiomodulation effects with violet-blue and red light in experimental oncogenesis

Introduction. There is still an open question about the limits of medical use of low-intensity electromagnetic radiation of the optical range in tumor growth due to the risk of increased proliferation of tumor cells. The conditions under which the tumor process is stimulated, as well as the mechanisms of photobiomodulation in oncological pathology, remain unclear. The aim of the study - in vitro evaluation and comparison of the effect of low-intensity electromagnetic radiation with wavelengths of 400, 460 and 660 nm on the activity of free-radical processes in tumor tissue and blood in normal and growing experimental neoplasia. Materials and methods. The study was conducted on biological material obtained from white non-linear rats intact and with subcutaneously transplanted cholangiocellular cancer MS-1. The sources of low-intensity radiation with wavelengths of 400, 460 and 660 nm were led generators. The content of hemoglobin, the activity of superoxide dismutase and catalase, changes in the overall level of free radical processes and antioxidant activity by induced chemiluminescence, and DNA damage by the method of DNA comets were studied. Data analysis was performed using nonparametric statistics methods. Results. The multidirectional effect of radiation with wavelengths of 400, 460 and 660 nm on free-radical homeostasis indicators at the early and late stages of tumor growth, as well as the dependence of biological effects on the wavelength of radiation, was found. Conclusions. The results obtained allow making a number of assumptions about the mechanisms of action of the optical electromagnetic waves on tumor growth, modulating free radical processes in the tumor-bearing organism.

Antiangiogenic therapy with Nintedanib affects hypoxia, angiogenesis and apoptosis in the ventral prostate of TRAMP animals

The antiangiogenic therapy for prostate cancer with Nintedanib, a potent inhibitor of important growth factor receptors, has been proven to delay tumor progression and arrest tumor growth; thus, the aim herein is to evaluate Nintedanib effects on tumor cells, besidesangiogenesis and apoptosis processes, metalloproteinases and hypoxia factor in an animal model. Nintedanib promoted growth inhibition and cell death in a dose-dependent manner, showing no tumor selectivity. Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) were treated with Nintedanib (10mg/kg/day) in different stages of tumor development and the ventral prostate was examined for protein levels by means of immunohistochemistry and Western blotting and apoptosis evaluation. In vitro antiproliferative activity of Nintedanib was also assessed in nine human tumor cell lines. Early Nintedanib treatment has shown decreased levels of FGF-2, VEGFR-1, MMP-9 and HIF-1α and a significantly increased apoptosis of epithelial cells. Furthermore, late Nintedanib treatment decreased FGF-2, VEGFR-1 and FGFR-3 levels. Importantly, even after treatment discontinuation, treated animals displayed a significant decrease in VEGFR-1 as well as MMP-9. Although Nintedanib treatment in late stages of tumor growth has shown some good results, it is noteworthy that the drug presents the best tissue response when administered in the early stages of disease development. Nintedanib treatment has shown to be a promising approach for prostate cancer therapy, especially in the early stages of the disease, interfering in different carcinogenesis progression pathways.

Oncogenic MAGEA-TRIM28 ubiquitin ligase downregulates autophagy by ubiquitinating and degrading AMPK in cancer

Autophagy is commonly altered in cancer and has a complicated, but important role in regulation of tumor growth. Autophagy is often tumor suppressive in the early stages of cancer development, but contributes to the late stages of tumor growth. Because of this, putative oncogenes that modulate autophagy signaling are especially interesting. Here we discuss our recent work detailing the function of the MAGEA-TRIM28 ubiquitin ligase as an oncogene product that targets PRKAA1/AMPKα1 for ubiquitination and proteasome-mediated degradation. Degradation of AMPK, a master cellular energy sensor and regulator, by MAGEA-TRIM28 results in significantly reduced autophagy and changes in cellular metabolism, including upregulation of MTOR signaling. Overall, expression of MAGEA3 (or MAGEA6) and degradation of AMPK is sufficient to induce transformation of normal cells and promote multiple hallmarks of cancer.

Immune cell responses to combined treatment of murine melanoma with ALT-803 and Sunitinib in normal and chronic alcohol consuming mice (TUM2P.1032)

Abstract The immune enhancing IL-15 super agonist ALT-803 and Sunitinib, a tyrosine kinase inhibitor that inhibits immunosuppressive myeloid derived suppressor cells (MDSC) and T regulatory (Treg) cells, were examined for their single and combined effects on immune cell phenotype in normal and chronic alcohol consuming (CAC) mice inoculated with s.c. B16BL6 melanoma. ALT-803 alone and in combination with Sunitinib initially increased CD8+CD44hi and CD8+CD44hiCD62Llo memory phenotype T cells in the spleen and PBL. IFN-γ producing CD8+CD44hi T cells increased in the spleen and the increase was more robust in CAC versus normal mice. These responses decayed during the later stages of tumor growth, and declined more rapidly in the CAC mice. Sunitinib alone did not affect CD8+ T cells or their IFN-γ production. Sunitinib alone and in combination with ALT-803 prevented the increase in CD11b+Gr-1int MDSC in the spleen associated with no treatment or ALT-803 treatment in normal mice; however, the effect was less pronounced in CAC mice where MDSC did not increase in the untreated group over time. MDSC in the PBL increased over time in untreated and ALT-803-treated normal mice. Sunitinib prevented this increase until the late stages of tumor progression. Sunitinib alone and in combination suppressed CD4+CD25+FoxP3+ Treg cells in the spleen during tumor progression.

Complete remission of cancer in late-stage disease by radiation and transfer of allogeneic MHC-matched immune T cells: lessons from GvL studies in animals.

Most immunotherapy studies in animal tumor models are performed in early stages of the disease. Reports on the studies of treatment in late stages of tumor growth and metastasis are much rarer. To guide future efforts for treatment in late-stage disease, a model of effective immune rejection of advanced metastasized cancer is reviewed and lessons therefrom are summarized. Already cachectic DBA/2 mice with a subcutaneously transplanted syngeneic tumor (ESb-MP lymphoma) of 1.5 cm diameter and with macroscopic liver and kidney metastases at 4 weeks could be successfully treated by a combination of sublethal (5 Gy) irradiation followed by a single transfer of 20 million anti-tumor immune spleen cells from tumor-resistant allogeneic MHC-B10.D2 mice. Following intravenous cell transfer, the primary tumors became encapsulated and were eventually rejected from the skin while visceral metastases gradually disappeared leaving behind only scar tissue. There was wound-healing at the site of the rejected primary tumor, and the animals survived long term without any tumor recurrence. The complete eradication of late-stage disease by adoptive cellular immunotherapy could be corroborated noninvasively by (31)P-NMR spectroscopy of primary tumors and by (1)H-NMR microimaging of liver metastases. Conclusions from functional mechanistic studies in this model are summarized and clinical implications discussed.

Bimodal role of Kupffer cells during colorectal cancer liver metastasis

Kupffer cells (KCs) are resident liver macrophages that play a crucial role in liver homeostasis and in the pathogenesis of liver disease. Evidence suggests KCs have both stimulatory and inhibitory functions during tumor development but the extent of these functions remains to be defined. Using KC depletion studies in an orthotopic murine model of colorectal cancer (CRC) liver metastases we demonstrated the bimodal role of KCs in determining tumor growth. KC depletion with gadolinium chloride before tumor induction was associated with an increased tumor burden during the exponential growth phase. In contrast, KC depletion at the late stage of tumor growth (day 18) decreased liver tumor load compared with non-depleted animals. This suggests KCs exhibit an early inhibitory and a later stimulatory effect. These two opposing functions were associated with changes in iNOS and VEGF expression as well as T-cell infiltration. KC depletion at day 18 increased numbers of CD3+ T cells and iNOS-expressing infiltrating cells in the tumor, but decreased the number of VEGF-expressing infiltrating cells. These alterations may be responsible for the observed reduction in tumor burden following depletion of pro-tumor KCs at the late stage of metastatic growth. Taken together, our results indicate that the bimodal role of KC activity in liver tumors may provide the key to timing immunomodulatory intervention for the treatment of CRC liver metastases.

Prognostic relationship of metabolic profile obtained of melanoma B16F10

Melanoma is a type of cancer that reaches more people in the world, characterized by genetic mutations that trigger the growth of disorganized cells. The diagnosis of skin tumors by invasive techniques has become a risk to the patients, so the search for new non-invasive techniques has been the subject of research in recent years. The objective of this work is to propose a non-invasive method prognosis based on the identification of specific biomarkers of the cancer, known as metabolomics analysis. For this study, we used B16F10 melanoma tumor cells and metabolic profiles were obtained at three time-periods by 1HNMR and comparison with the cell cycle, apoptosis pathways and proliferation index. The metabolic profiles show the relationship between the metabolites found with energy metabolism, pathways of apoptosis and proliferation, which showed increases in proportion during growth and progression. Were found 29 metabolites, of which the differentially expressed are: lactate, aspartate, glycerol, lipids, alanine, myo-inositol, phosphocholine, choline, acetate, creatine and taurine. Choline and creatine are closely related with tumor progression, and are inversely expressed in later stages of tumor growth, which demonstrates the ability to be markers of tumor progression or monitoring the pharmacological efficacy when combined with other therapies. We conclude that the metabolome appeared as effective non-invasive technique predicts, besides providing possible biomarkers of melanoma.

A novel iNKT cell-mediated vaccine maneuver induces potent long-lasting anti-tumor immunity for advanced prostate cancer by boosting CD8+T cell memory and function (53.24)

Abstract iNKT cells have considerable therapeutic promise in cancer, but so far this has not been fully realized. At issue has been delivering iNKT ligand and tumor Ag to the same APC for optimal processing and presentation. Also, although tumor-infiltrating APCs express CD1d in both prostate cancer (PCa) models and patients, tumor induced tolerance and suppression leads to defective iNKT activation. Whole-cell tumor vaccines represent a promising treatment approach and prevent tumor progression in multiple models of cancer. Here, we use an optimized approach consisting of alpha-galactosylceramide (aGalCer) and the GM-CSF-based PCa vaccine, ‘GVax’ combined with IL-12. This approach was capable of breaking tumor-induced tolerance and causing dramatic and substantial tumor regression and survival in large advanced prostate tumors in TRAMP mice. Remarkably, 10/12 vaccinated >9 month old mice rejected tumors, 'breaking tolerance', even at this late stage of tumor growth. The vaccine induced strong immune responses capable of reversing INKT defects in tumor-bearing mice as well as in patient iNKT in vitro. Both IL-12 and aGalCer were crucial for this response. Vaccinated mice had higher levels and activation of tumor-specific CD8 T, CD4 T and iNKT cells, and greater memory CD8 T cell expansion capable of producing the effector cytokines IFN-γ and TNF-α in response to tumor. These studies shed light on how whole cell vaccines targeting iNKTs can be utilized in PCa immunotherapy.

Abstract 3475: Tumor stage dependent contribution of bone marrow derived cells to tumor neo-vascularization

Abstract Introduction: The role of bone marrow derived cells (BMDC) in tumor neo-vascularization remains controversial. We have previously demonstrated that BMDC contribute to tumor neovascularization by differentiating into vascular support cells and not endothelial cell. In this study we focus on establishing the spatiotemporal role of BMDC in tumor neovascularisation, focusing on whether it is a tumor growth stage dependent process and more specifically examining the patterns of BMDC integration into tumor vasculature in response to radiation therapy (RTx). Methods: Animal Models: Bone marrow of NOD/SCID mice were stably reconstituted with BM harvested from green-fluorescent protein (GFP) transgenic mice. Intracranial (ic) xenografts were generated in intracranial window models (ICW) using a panel of glioma cell and breast cancer cells stably expressing mCherry. In-vivo imaging: Two-photon laser capture microscopy was used to obtain high-resolution real-time in-vivo longitudinal images of the tumor cells, tumor vasculature and tracing of the circulating GFP+BM cells. Mice were imaged 1d,2d,3d,7d,10d,14d,21d & 30d following cell implantation or RTx. Brain Tumor Analysis: Mice were sacrificed using perfusion fixation and brains collected for correlative immunohistochemical and Immunofluorscent confocal analysis. RTx: Using in-house designed x-ray micro-irradiator stereotactic radiation was delivered through ICW to ic-xenografts, treated with either 3×2Gy or 3×5Gy. Tumor+RTx was compared to RTx alone or tumor alone (n=15 each arm). Results: At early stages of tumor formation (<1wk) there is 90% integration of BMDC into the tumor vasculature, while to late stages of tumor growth (>2 wks) only 10% integration of BMDC is seen. At later stages of tumor growth (>2 wks) BMDC incorporation in the tumor vasculature is predominantly seen in the tumor periphery compared to the tumor centre, with the peripheral vessels demonstrating almost 100% integration of BMDC compared to the centre of the tumor where the vasculature have no integration of BMDC. Conclusions: Our results are the first to demonstrate that there is a specific tumor growth stage dependent contribution of BMDC to tumor neo-vascularization. At early stages of tumor growth neo-ascularization relies on host BMDC, while at later stages of tumor growth vascularization relies on branching of pre-existing vessels. A significant difference in extent of BMDC integration is noted between periphery versus the centre of the tumor, suggesting that the internal tumor micro-environment regulates neoascularization differently from the periphery. Intriguingly, this distinction is not seen with tumor neovascularization in recurrent tumors following RTx. These results have significant therapeutic implications and point to a need for scheduling of anti-angiogenic therapies according to tumor growth stage and timing post RTx. 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 3475. doi:10.1158/1538-7445.AM2011-3475

Abstract 481: In vivo imaging identifies that myf5+ embryonal rhabdomyosarcoma-propagating cells are dynamically reorganized during tumor growth

Abstract Embryonal rhabdomyosarcoma (ERMS) is a devastating pediatric cancer with specific features of muscle differentiation. Here, we use a transgenic zebrafish model of RAS-induced ERMS and show that myf5+ ERMS cells possess self-renewal potential, confirming that ERMS is a cancer stem cell driven disease. The ERMS-initiating cell is molecularly similar to an activated satellite cell and expresses myf5, m-cadherin, and c-met. To directly visualize tumor-initiating cells in vivo, fluorescent transgenic zebrafish were created that express myf5-GFP, myogenin-h2b-mRFP, and mylz2-lyn-cyan. These transcription factors are expressed during distinct stages of muscle/ERMS development and transgenic reporter lines using these gene promoters allow self-renewing cells to imaged in live animals by confocal microscopy. myf5-GFP+ ERMS-initiating cells from early stage tumors lie in immediate proximity to muscle fibers where normal satellite cells reside. However, as tumors grow, the niche is greatly expanded with the number of myf5-GFP+ cells increasing by 10-fold per fiber. By late stages of tumor growth, myf5-GFP+ ERMS cells are re-localized into a new tumor-specific stem cell niche that is no longer associated with fibers and is separated from more differentiated myogenin+ and myogenein/mylz2 double positive tumor cells. Analysis of human ERMS showed that tumors also contain regional niches of differentiated and undifferentiated cells. Time-lapse imaging of live zebrafish showed that myf5-GFP+ ERMS cells move only locally within the tumor and remain largely confined to stable self-renewing niches. By contrast, myogenin+ cells move dynamically throughout tumors and colonize new areas for tumor growth by both migration through the tissue and entry into the vasculature system. Following colonization by myogenin+ cells, self-renewing myf5+ cells are recruited to new areas of tumor growth. Our findings reconcile the clinical observation that myogenin-positivity in RMS correlates with poor outcome, because it is these cells that intravasate into blood vessels, invade new areas of tumor growth, and recruit slow moving self-renewing cells to newly colonized areas. Taken together, our in vivo imaging studies provide unprecedented access to directly visualize tumor growth in real-time and to define the kinetics of self-renewal and tumor dissemination as has never been assessed in any experimental model. 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 481. doi:10.1158/1538-7445.AM2011-481

PARP1 is activated at telomeres upon G4 stabilization: possible target for telomere-based therapy

New anti-telomere strategies represent important goals for the development of selective cancer therapies. In this study, we reported that uncapped telomeres, resulting from pharmacological stabilization of quadruplex DNA by RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate), trigger specific recruitment and activation of poly-adenosine diphosphate (ADP) ribose polymerase I (PARP1) at the telomeres, forming several ADP-ribose polymers that co-localize with the telomeric repeat binding factor 1 protein and are inhibited by selective PARP(s) inhibitors or PARP1-specific small interfering RNAs. The knockdown of PARP1 prevents repairing of RHPS4-induced telomere DNA breaks, leading to increases in chromosome abnormalities and eventually to the inhibition of tumor cell growth both in vitro and in xenografts. More interestingly, the integration of a TOPO1 inhibitor on the combination treatment proved to have a high therapeutic efficacy ensuing a complete regression of the tumor as well as a significant increase in overall survival and cure of mice even when treatments started at a very late stage of tumor growth. Overall, this work reveals the unexplored link between the PARP1 and G-quadruplex ligands and demonstrates the excellent efficacy of a multi-component strategy based on the use of PARP inhibitors in telomere-based therapy.

Abstract C170: Inhibition of poly(ADP-ribose) polymerase (PARP) increases the therapeutic efficacy of the G-quadruplex ligand RHPS4/irinotecan combination in cancer xenografts

Abstract Background and Aim: The G-quadruplex ligand RHPS4 is known to induce a rapid activation of DNA damage response at telomeres and poly (ADP-ribosyl)ation is involved in the regulation of many cellular processes such as DNA damage repair. We recently showed that the combination of RHPS4 with camptothecins has a strong synergistic interaction in vitro on colon cancer lines and produced a marked antitumor activity on xenografts. Unfortunately, this combination failed to cure animals as no complete remissions were reported. So, based on the observation that PARP inhibitors sensitize tumor cells to camptothecins and in the attempt to search for a more effective anticancer therapy, we evaluated a multicomponent strategy based on the addition of the PARP inhibitor GPI 15427 to the previously established camptothecins/ RHPS4 combination. Material and Methods: PARP activity and poly(ADP-ribose) formation have been evaluated in cells upon RHPS4 treatment. Moreover, the presence of poly(ADP-ribose) at the telomeres has been investigated by coimmunofluorescence experiments using an anti-TRF1 antibody to mark telomeres. The involvement of PARP activity in the cellular response to RHPS4 has been studied by treatment with the PARP inhibitor GPI 15427 followed by analysis of the formation of TIFs (Telomeres Disfunction Induced Foci) and evaluation of the clonogenic ability of the human colon carcinoma cells line HT29. In vivo experiments were performed on HT29 xenografts to evaluate the therapeutic efficacy of GPI 15427/Irinotecan/RHPS4 combination. Results and Conclusions: RHPS4 produced a rapid induction of PARP activity and, interestingly, most of the ADP-ribose polymers induced by RHPS4 were localized at telomeres. Consistently with these results, the PARP-1 protein was found to be localized at telomeres upon drug treatment, thus providing a rational for RHPS4 and PARP inhibitors combination. Indeed, RHPS4/GPI 15427 combination strongly increased the number of TIFs/cell and reduced the survival of HT29 cells compared to the treatment with RHPS4 alone. The in vivo experiments confirmed these results and demonstrated the high efficacy of GPI 15427/Irinotecan/RHPS4 combination as this multicomponent strategy produced an impressive inhibition of tumor weight and a marked tumor growth delay. Notably, after the triple combination a complete regression of tumors was observed in most of the mice treated accompanied by a significant increase of overall survival. Finally, a complete cure of about 50% of mice was observed. Interestingly, this treatment remained effective even when started at a very late stage of tumor growth. In conclusion, these data suggest that the integration of a PARP inhibitor with Irinotecan/RHPS4 combination could be a promising strategy to improve the response of solid tumors to antitumoral agents. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C170.

Multi-scale models of cell and tissue dynamics

Cell and tissue movement are essential processes at various stages in the life cycle of most organisms. The early development of multi-cellular organisms involves individual and collective cell movement; leukocytes must migrate towards sites of infection as part of the immune response; and in cancer, directed movement is involved in invasion and metastasis. The forces needed to drive movement arise from actin polymerization, molecular motors and other processes, but understanding the cell- or tissue-level organization of these processes that is needed to produce the forces necessary for directed movement at the appropriate point in the cell or tissue is a major challenge. In this paper, we present three models that deal with the mechanics of cells and tissues: a model of an arbitrarily deformable single cell, a discrete model of the onset of tumour growth in which each cell is treated individually, and a hybrid continuum-discrete model of the later stages of tumour growth. While the models are different in scope, their underlying mechanical and mathematical principles are similar and can be applied to a variety of biological systems.

Host-Derived Angiopoietin-2 Affects Early Stages of Tumor Development and Vessel Maturation but Is Dispensable for Later Stages of Tumor Growth

The angiopoietin/Tie2 system has been identified as the second vascular-specific receptor tyrosine kinase system controlling vessel assembly, maturation, and quiescence. Angiopoietin-2 (Ang-2) is prominently up-regulated in the host-derived vasculature of most tumors, making it an attractive candidate for antiangiogenic intervention. Yet, the net outcome of Ang-2 functions on tumor angiogenesis is believed to be contextual depending on the local cytokine milieu. Correspondingly, Ang-2 manipulatory therapies have been shown to exert protumorigenic as well as antitumorigenic effects. To clarify the role of Ang-2 for angiogenesis and tumor growth in a definite genetic experimental setting, the present study was aimed at comparatively studying the growth of different tumors in wild-type and Ang-2-deficient mice. Lewis lung carcinomas, MT-ret melanomas, and B16F10 melanomas all grew slower in Ang-2-deficient mice. Yet, tumor growth in wild-type and Ang-2-deficient mice dissociated during early stages of tumor development, whereas tumor growth rates during later stages of primary tumor progression were similar. Analysis of the intratumoral vascular architecture revealed no major differences in microvessel density and perfusion characteristics. However, diameters of intratumoral microvessels were smaller in tumors grown in Ang-2-deficient mice, and the vasculature had an altered pattern of pericyte recruitment and maturation. Ang-2-deficient tumor vessels had higher pericyte coverage indices. Recruited pericytes were desmin and NG2 positive and predominately alpha-smooth muscle actin negative, indicative of a more mature pericyte phenotype. Collectively, the experiments define the role of Ang-2 during tumor angiogenesis and establish a better rationale for combination therapies involving Ang-2 manipulatory therapies.