Tumor Cell Compartment Research Articles

Abstract 1564: Investigating the interaction of tumor xenografts with their microenvironment by combining human- and mouse- specific assays with high throughput RT-qPCR

Abstract Tumour growth is governed by the interaction with its microenvironment through pathways that regulate tumour cell function, angiogenesis, and recruitment of associated stromal cells and immune infiltrate. These complicated biological networks can be studied in tumour xenografts using a combination of gene expression, protein and histological analysis. The majority of gene expression studies either probe expression of specific transcripts by RT-qPCR, or use broad Affymetrix microarrays to examine 1000s of genes. RT-PCR experiments may be too focussed studying the interaction of a small number of pathways, while Affymetrix is often not sensitive enough to detect low abundance transcripts, or is limited by species specificity of the probe sets. For example studying tumour stromal and endothelial cells can be challenging because these cells comprise only a small proportion of the tumour. In this study we have used high throughput RT-qPCR to specifically analyse 180 human and mouse genes (or orthologues) thought to regulate key stromal/tumour interactions in a broad xenograft panel. This approach enabled us to determine the expression patterns of potential angiogenic, migratory and survival drives of a diverse range of tumour xenograft models. Across the xenograft panel these genes distinguished tumours with an Epithelial and Mesenchymal phenotype. Interestingly we find that the transcript profile of the supporting stroma (all murine transcripts) is similar between models and key genes cluster with physiological characteristics such vessel, stromal or inflammatory phenotypes. We also used this approach to assess pharmacodynamic changes in gene expression in the tumour and host compartments in response to a VEGF signalling inhibitor. Surprisingly there was little modulation in the expression of genes in the tumour cell compartment. However, there was a clear down regulation of transcripts corresponding to endothelial cells with a concomitant reduction in the tumour vasculature. This approach has enabled us to gain insight into pre-clinical models and the response to a host targetted therapy. The data provides information that enhances understanding that can be derived using histological analysis and standard protein expression. 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 1564. doi:10.1158/1538-7445.AM2011-1564

Double Attack on Tumors by Targeting With Guidance Molecules

Therapies targeting vascular endothelial growth factor and its receptors have become part of standard tumor therapy for most forms of cancer within the last 7 years. However, it is increasingly recognized that the efficacy of such therapies is limited and transient. In fact, the limited clinical efficacy of established antiangiogenic tumor therapies has raised the concern that the moderate gain in cancer patient care may not justify the significant societal investment in such therapies. In turn, it needs to be considered that the first generation of approved antivascular endothelial growth factor/vascular endothelial growth factor receptor drugs mark only the beginning of a new era of antitumor therapy that goes beyond classical direct tumor cell targeting strategies to attack the nonneoplastically transformed tumor-associated stroma. Clearly, the development of antistroma therapies is only at its beginning, and the hunt for additional targets to interfere with tumor progression concentrates particularly on molecules that may affect both the tumor cell compartment and tumor-associated stromal cell mechanisms. See accompanying article on page 741 Guidance molecules, such as the semaphorins, may …

Transcription factors zeb1, twist and snai1 in breast carcinoma

BackgroundEpitheliomesenchymal transition (EMT) is the process where cancer cells attain fibroblastic features and are thus able to invade neighboring tissues. Transcriptional factors zeb1, snai1 and twist regulate EMT.MethodsWe used immunohistochemistry to investigate the expression of zeb1, twist and snai1 in tumor and stromal compartments by in a large set of breast carcinomas. The results were compared with estrogen and progesterone receptor status, HER2 amplification, grade, histology, TNM status and survival of the patients.ResultsNuclear expression for twist was seen in the epithelial tumor cell compartment in 3.6% and for snai1 in 3.1% of the cases while zeb1 was not detected at all in these areas. In contrast, the tumor stromal compartment showed nuclear zeb1 and twist expression in 75% and 52.4% of the cases, respectively. Although rare, nuclear expression of twist in the epithelial tumor cell compartment was associated with a poor outcome of the patients (p = 0.054 log rank, p = 0.013, Breslow, p = 0.025 Tarone-Ware). Expression of snai1, or expression of zeb1 or twist in the stromal compartment did not have any prognostic significance. Furthermore, none of these factors associated with the size of the tumors, nor with the presence of axillary or distant metastases. Expression of zeb1 and twist in the stromal compartment was positively associated with a positive estrogen or progesterone receptor status of the tumors. Stromal zeb1 expression was significantly lower in ductal in situ carcinomas than in invasive carcinomas (p = 0.020). Medullary carcinomas (p = 0.017) and mucinous carcinomas (p = 0.009) had a lower stromal expression of zeb1 than ductal carcinomas. Stromal twist expression was also lower in mucinous (p = 0.017) than in ductal carcinomas.ConclusionsExpression of transcriptional factors zeb1 and twist mainly occur in the stromal compartment of breast carcinomas, possibly representing two populations of cells; EMT transformed neoplastic cells and stromal fibroblastic cells undergoing activation of zeb1 and twist due to growth factors produced by the tumor. However, epithelial expression of twist was associated with a poor prognosis, hinting at its importance in the spread of breast carcinoma.

Cancer evolution and individual susceptibility

Cancer susceptibility is due to interactions between inherited genetic factors and exposure to environmental carcinogens. The genetic component is constituted mainly by weakly acting low-penetrance genetic variants that interact among themselves, as well as with the environment. These low susceptibility genes can be categorized into two main groups: one includes those that control intrinsic tumor cell activities (i.e. apoptosis, proliferation or DNA repair), and the other contains those that modulate the function of extrinsic tumor cell compartments (i.e. stroma, angiogenesis, or endocrine and immune systems). Genome-Wide Association Studies (GWAS) of human populations have identified numerous genetic loci linked with cancer risk and behavior, but nevertheless the major component of cancer heritability remains to be explained. One reason may be that GWAS cannot readily capture gene-gene or gene-environment interactions. Mouse model approaches offer an alternative or complementary strategy, because of our ability to control both the genetic and environmental components of risk. Recently developed genetic tools, including high-throughput technologies such as SNP, CGH and gene expression microarrays, have led to more powerful strategies for refining quantitative trait loci (QTL) and identifying the critical genes. In particular, the cross-species approaches will help to refine locations of QTLs, and reveal their genetic and environmental interactions. The identification of human tumor susceptibility genes and discovery of their roles in carcinogenesis will ultimately be important for the development of methods for prediction of risk, diagnosis, prevention and therapy for human cancers.

Endoglin expression in breast tumor cells suppresses invasion and metastasis and correlates with improved clinical outcome

Tumor growth factor-β (TGF-β) signaling in cancer has been implicated in growth suppression of early lesions and enhancing tumor cell invasion and metastasis. However, the cellular mechanisms that determine this signaling output in individual tumors are still largely unknown. In endothelial cells, TGF-β signaling is modulated by the TGF-β co-receptor endoglin (CD105). Here we demonstrate that endoglin is expressed in a subset of invasive breast cancers and cell lines and is subject to epigenetic silencing by gene methylation. Endoglin downregulation in non-tumorigenic MCF10A breast cells leads to the formation of abnormal acini in 3D culture, but does not promote cell migration or transformation. In contrast, in the presence of activated ErbB2, endoglin downregulation in MCF10A cells leads to enhanced invasion into a 3D matrix. Consistent with these data, ectopic expression of endoglin in MDA-MB-231 cells blocks TGF-β-enhanced cell motility and invasion and reduces lung colonization in an in vivo metastasis model. Unlike endothelial cells, endoglin does not modulate Smad-mediated TGF-β signaling in breast cells but attenuates the cytoskeletal remodeling to impair cell migration and invasion. Importantly, in a large cohort of invasive breast cancers, lack of endoglin expression in the tumor cell compartment correlates with ENG gene methylation and poor clinical outcome.

Abstract 539: Endosialin (TEM1) is a tumor stroma marker and receptor for the metastasis-related Mac-2 binding protein

Abstract Endosialin is a heavily sialylated C-type lectin. It is a single transmembrane molecule that was identified by two independent studies as an angiogenic endothelial specific marker. However, recent high resolution confocal expression studies by our and other laboratories have revealed that Endosialin is not expressed by endothelial cells but instead by angiogenic blood vessel associated pericytes. Detailed tissue array-based expression profiling validated Endosialin as an oncofetal gene product of the mesenchymal lineage that is hardly detectable in normal tissues but abundantly expressed by tumor vessel-associated pericytes and tumor stromal fibroblasts (myofibroblasts). As such, Endosialin was not just identified as a marker of activated pericytes but based on its exclusive tumor-associated expression also as a novel therapeutic tumor stroma target. Cytokine induction experiments identified Endosialin as a major downstream target of TGFß stimulation. Cellular siRNA-based loss-of-function experiments unravelled a role of Endosialin in the regulation of mesenchymal cell adhesion, migration and proliferation. These findings prompted experiments aimed at identifying the extracellular ligand of Endosialin. Using Endosialin-Fc as an affinity probe, the metastasis associated molecule Mac-2BP/90K was identified as high affinity extracellular Endosialin ligand. As Endosialin, Mac-2BP/90K displays an exclusive tumor progression associated expression pattern. However, in contrast to the stromal expression of Endosialin, Mac-2BP/90K is strictly expressed by the tumor cell compartment. The mutually exclusive expression pattern suggests a repulsive interaction between Endosialin and Mac-2BP/90K which was confirmed in cellular experiments. Ongoing experiments are aimed at mechanistically studying the role of the Endosialin - Mac-2BP/90K interaction in controlling the crosstalk between the tumor cell and the stromal cell compartments during tumor progression and metastasis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 539.

Metronomic administration of the drug GMX1777, a cellular NAD synthesis inhibitor, results in neuroblastoma regression and vessel maturation without inducing drug resistance

High-risk neuroblastoma is a rapidly growing tumor with a survival rate below 50%. A new treatment strategy is to administer chemotherapeutic drugs metronomically, i.e., at lower doses and frequent intervals. The aim of the study was to investigate the effects of GMX1777, a chemotherapeutic drug affecting cellular energy metabolism, in a mouse model for high-risk neuroblastoma. Female SCID mice were injected s.c. with MYCN-amplified human neuroblastoma cells and randomized to either treatment with GMX1777 or vehicle. In some animals, treatment was discontinued allowing tumor relapse. Treatment response was evaluated using the pediatric preclinical testing program (PPTP). Immunohistochemistry and qRT-PCR was performed on tumor cryosections to investigate the microscopic and molecular changes in tumors in response to GMX1777. Despite an increase in vessel density, tumor regression and a high group response score according to PPTP criteria was induced by GMX1777 without inducing drug resistance. Treatment resulted in inhibition of tumor cell proliferation, vessel maturation, reduced hypoxia, increased infiltration of MHC class II negative macrophages and expansion of the nonvascular stromal compartment. Decreased stromal VEGF-A and PDGF-B mRNA in response to treatment together with the structural data suggest a "deactivation" or "silencing" of the tumor stroma as a paracrine entity. In conclusion, GMX1777 was highly efficient against high-risk neuroblastoma xenografts through modulation of both the tumor cell and stromal compartment.

Highlights of This Issue

In an effort to target the tumor vasculature, metronomic approaches to deliver chemotherapy seek to deliver small doses frequently to avoid toxicities of intermittent doses at the maximum tolerated dose (MTD). Pioneered in the laboratory of the late Dr. Judah Folkman, applying this approach to humans has offered promise, but how to achieve the maximal value of this approach in the clinic has remained uncertain. Complementary preclinical papers by Hashimoto and colleagues and Merritt and colleagues offer an important opportunity to apply this strategy in ovarian cancer. An oral formulation of topotecan, an anti-topoisomerase I cytotoxic in combination with pazopanib, a recently approved VEGF-R kinase antagonist, caused striking suppression of ovarian carcinoma xenograft growth. Importantly, the ovarian carcinoma cells in the Merritt paper expressed VEGF-R in the tumor cells. Thus, this metronomic approach when applied to human ovarian cancer could cause beneficial effects in both the tumor and endothelial cell compartment. These findings are “low hanging fruit” for clinical trial development, given the known activity of topotecan as a single agent in MTD doses in ovarian cancer, albeit with toxicity that might be avoidable if combined with pazopanib on a metronomic schedule.In-transit metastatic melanoma typically presents as multifocal lesions, providing a unique setting for evaluating the utility of gene expression profiling in defining patient-tailored regional therapeutic strategies as well as assessing treatment efficacy. Augustine and colleagues evaluated 55 in-transit melanoma lesions across 29 patients and showed that, within a patient, lesions harbor concordant patterns of gene expression. Their results demonstrate that a single in-transit lesion is representative of residual tumor burden in terms of gene expression signatures predictive of response to therapy and can be used to characterize the biology of a patient's in-transit disease.Six members of the peroxiredoxin (PRDX) family of thiol-dependent peroxidases have been identified in mammalian tissues. Among them, PRDX6 has a unique property; it possesses phospholipase A2 (PLA2) activity in addition to peroxidase and, thus, is considered a bifunctional enzyme. However, the physiological role of its PLA2 activity is not determined yet. In this study, Ho and colleagues analyzed functions of PRDX6 in lung cancer cells. This study is the first to define the functions of PRDX6's enzymatic activities in metastasis and to show arachidonic acid's involvement in PRDX6 action in intact cells. These novel findings provide a significant step towards elucidating the role of PRDX6 in cancer and the mechanism of its action.Nutlin-3a is a small molecule MDM2 antagonist that stabilizes p53 by blocking the interaction between p53 and MDM2. Numerous studies have supported the potential clinical use of Nutlin-3a as an anticancer agent, based on the potent ability of Nutlin-3a to promote growth arrest or apoptosis in p53-wild-type cancer cells. In the study by Moran and Maki, it was found that Nutlin-3a treatment caused a cytoskeletal rearrangement in multiple p53 wild-type cancer cell lines. The results indicate that clinical use of Nutlin-3a may inhibit directional migration and have antimetastatic potential against p53 wild-type cancers, in addition to its already established antiproliferative effects.

Antitumor Activity of Targeting Src Kinases in Endothelial and Myeloid Cell Compartments of the Tumor Microenvironment

Several Src family kinase (SFK) inhibitors have entered clinical trials based on their direct effects against tumor cells. Here, we characterize the effects of targeting Src kinases on the tumor microenvironment and how these effects influence tumor growth. Human cancer cells grown in cell culture or in mice were treated with dasatinib, a small-molecule inhibitor of SFKs. Tumor cell, endothelial cell, and myeloid cell compartments within the tumor microenvironment were analyzed. Primary human endothelial cells and freshly isolated CD11b+/CD11c- myeloid cells from mice were treated with dasatinib in cell culture. Cellular functions and signaling pathways affected by dasatinib were evaluated. Dasatinib was not cytotoxic in cell culture against the human cancer cell lines investigated here. However, dasatinib administration in human tumor-bearing mice suppressed tumor growth associated with increased tumor cell apoptosis, decreased microvessel density, and reduced intratumoral CD11b+ myeloid cells. Dasatinib directly inhibited motility and other functions of endothelial and myeloid cells, accompanied by the inhibition of phosphorylation of SFKs and downstream signaling. Tumor-infiltrating myeloid cells were identified as the major source of matrix metalloproteinase (MMP)-9 in the tumor microenvironment. Dasatinib treatment reduced MMP-9 levels in the tumor microenvironment through the simultaneous inhibition of recruitment of MMP9+ myeloid cells and MMP-9 gene expression in tumor-infiltrating myeloid cells. These findings suggest that Src kinase inhibitors such as dasatinib possess a previously unrecognized anticancer mechanism of action by targeting both host-derived endothelial and myeloid cell compartments within the tumor microenvironment.

The Calcineurin Inhibitor Tacrolimus Inhibits Breast Tumor Growth In Vivo.

Abstract Background: We recently reported a novel angiogenesis factor, secreted frizzle-related protein 2 (SFRP2), that stimulates angiogenesis via a calcineurin/ NFAT pathway and is highly expressed in human breast tumor endothelium (Courtwight et. al., Cancer Research 2009). NFAT is a transcription factor that mediates vascular development and VEGF and bFGF-induced angiogenesis. Tacrolimus (FK506) is a calcineurin inhibitor that inhibits NFAT activation and is FDA approved to prevent organ transplant rejection. Based on its mechanism of action, we hypothesized that tacrolimus would inhibit angiogenesis and breast tumor growth.Materials and Methods: Tumor growth in vivo: MMTV-neu transgenic mice were treated with tacrolimus 3 mg/kg/day i.p. for 21 days or control when tumors became palpable. Tumor volumes were measured with 3D ultrasound. Endothelial tube formation in vitro: ECMatrix was solidified into wells of a 96 well plate and endothelial cells were seeded onto the matrix at a concentration of 1x104 cells/well with SFRP2 7nM or VEGFA 60ng/ml. Tacrolimus 1µM or 1.5% DMSO control was added to the wells. The number of branch points were counted after 8 hours. MMTV-neu migration in vitro: Breast tumor cells were plated at 10,000 cells/well into a 96 well plate and allowed to become confluent. The cells were quiesced for 18 hours and tacrolimus (1µM) or 0.5% DMSO control was added to the wells and the wound was formed using a 1 ml pipette tip. Migration distance was measured with an ocular micrometer after 16 hours. Western blot analyses: Endothelial cells were treated for 1 hour with negative control, SFRP2 7nM, or SFRP2 7nM plus tacrolimus 10 µM. Nuclear protein was extracted and Western blot for NFATc3 was performed. Statistics: Means were compared with a two-tailed t-test.Results: Tumor growth in vivo: Tacrolimus inhibited the growth rate of MMTV-neu tumors by 49% (n=12 treated, n=9 controls, p=0.04), without signs of toxicity. Endothelial tube formation: Tacrolimus (1µM) inhibited SFRP2 induced endothelial tube formation by 64% (0.002, n=4), and VEGF induced tube formation by 69% (p=0.004, n=4). MMTV-neu tumor cell migration: Tacrolimus (1µM) inhibited MMTV-neu cell migration by 36% (p<0.05, n=4). Western blot for NFATc3: Treatment of endothelial cells with SFRP2 induced nuclear NFATc3 at 1 hour compared to control, and this increase was abolished by treatment with tacrolimus.Conclusion: The calcineurin inhibitor tacrolimus inhibits the growth rate of MMTV-neu transgenic tumors in vivo. The in vitro cellular effects of tacrolimus include inhibiting SFRP2 and VEGF-induced endothelial tube formation, abolishing SFRP2 activation of NFATc3 in endothelial cells, and inhibiting MMTV-neu cell migration, demonstrating a direct effect of tacrolimus on both the endothelial and tumor cell compartments. This establishes that NFAT may be a favorable therapeutic target to inhibit angiogenesis and breast cancer growth. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3170.

Expression of the Cancer Stem Cell Marker ALDH1A1 in Primary Breast Cancer: A Mechanism for Chemotherapy Resistance.

Abstract Background: Aldehyde dehydrogenase 1 (ALDH1) has been identified as a marker of breast cancer stem cells, and ALDH1 expression in primary breast tumors has been associated with resistance to chemotherapy and poor prognosis. ALDH1 is an enzyme that can detoxify reactive aldehyde compounds; however the mechanism of ALDH1 chemotherapy resistance and its promotion of the breast cancer stem cell phenotype is unclear. Materials and Methods: Illumina cDNA array was performed on primary breast tumors that were snap-frozen, and RNA was isolated from dense, viable tumor regions. Samples were normalized by rank-invariant method and gene expression was analyzed. Immunohistochemistry was performed for ALDH1A1 on initial and post-neoadjuvant tumor samples from patients who have undergone six cycles of chemotherapy. Clinical data including age, tumor pathology, lymph node status, and response to neoadjuvant therapy were available for analysis. Human breast cancer cell lines (MCF-7 and MDA-MB-468) were treated with increasing doses of retinoic acid (RA) with and without the retinoic acid receptor alpha antagonist, Ro 41-5253. Effects on cell proliferation and signal transduction pathways were assessed. Results: The expression of ALDH1A1 and/or ALDH1A3 isoform mRNA was found to be present in primary human breast tumors. The majority of these cases were noted as triple negative or Her-2 positive tumors. Evaluation of RA receptor expression revealed repressed RAR beta levels in all tumors, whereas RAR alpha expression was retained. Observations of patient tumor samples (pre and post neoadjuvant chemotherapy treatment) demonstrated high ALDH1A1 expression in tumor and stromal cell compartments. The expression was predominant in partial and non-responders to the neoadjuvant therapy. Interestingly, the tumor from a partial responder was converted to extremely high expresser of ALDH1A1 post-neoadjuvant therapy indicating a positive survival selection. Since ALDH1A1 can convert retinaldehyde to retinoic acid, breast cancer cell lines were treated with all-trans RA and responded with a significant decrease in cell proliferation. This effect was partially attenuated by pre-treatment with the RAR alpha antagonist, Ro 41-5253. Importantly, RA treatment of MCF-7 cells repressed MAPK signaling, which was also attenuated by pre-treatment with Ro 41-5253. Discussion: The observations from this study suggest that the expression of the breast cancer stem cell marker, ALDH1A1 on tumor cells or in tumor-associated stromal cells can provide a mechanism for survival of cancer during chemotherapy treatment. The conversion of therapeutic oxidant compounds to retinoic acid by ALDH1 may lead to increased RAR alpha signaling and suppression of proliferation. The suppression of proliferation correlates well with chemotherapy resistance in these neoadjuvant cases. This data demonstrates a possible mechanism for ALDH1 promotion of tumor cell survival under chemotherapy and suggests that treatment with RAR alpha antagonists may promote tumor cell susceptibility to current treatment modalities and improve the therapeutic window for primary, metastatic and chemo-refractory breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1135.

Vascular Endothelial Growth Factor Receptor-1 Is Synthetic Lethal to Aberrant β-Catenin Activation in Colon Cancer

PURPOSE: The Wnt/beta-catenin (beta-cat) signaling cascade is a key regulator of development, and dysregulation of Wnt/beta-cat contributes to selected cancers, such as colorectal, breast, and hepatocellular carcinoma, through abnormal activation of Wnt target genes. To identify novel modulators of the Wnt/beta-cat pathway that may emerge as therapeutic targets, we did an unbiased high-throughput RNA interference screen. EXPERIMENTAL DESIGN: A synthetic oligonucleotide small interfering RNA library targeting 691 known and predicted human kinases was screened in Wnt3a-stimulated human cells in a live cell luciferase assay for modulation of Wnt/beta-cat-dependent transcription. Follow-up studies of a selected high-confidence "hit" were conducted. RESULTS: A robust quartile-based statistical analysis and secondary screen yielded several kinases worthy of further investigation, including Cdc2L1, Lmtk3, Pank2, ErbB3, and, of note, vascular endothelial growth factor receptor (VEGFR)1/Flt1, a receptor tyrosine kinase (TK) with putative weak kinase activity conventionally believed to be a negative regulator of angiogenesis. A series of loss-of-function, genetic null, and VEGFR TK inhibitor assays further revealed that VEGFR1 is a positive regulator of Wnt signaling that functions in a glycogen synthase kinase-3beta (GSK3beta)-independent manner as a potential synthetic lethal target in Wnt/beta-cat-addicted colon carcinoma cells. CONCLUSIONS: This unanticipated non-endothelial link between VEGFR1 TK activity and Wnt/beta-cat signaling may refine our understanding of aberrant Wnt signaling in colon carcinoma and points to new combinatorial therapeutics targeted to the tumor cell compartment, rather than angiogenesis, in the context of colon cancer. (Clin Cancer Res 2009;15(24):7529-37).

Tumor Systems Need to be Rendered Usable for a New Action-Theoretical Abstraction: The Starting Point for Novel Therapeutic Options

Background: A tumor system not only consists of diverse cell types but also comprises all components of action insofar that these components are oriented in terms of diverse cell types. Methods: Thus, it is necessary to decode paradox situations of cellular rationalization, deformation, and communication processes or, in other words, to uncover inconsistencies within tumor cell compartments or distinct topologies of aggregated action effects. Here, a theory may be helpful that discharges into an action-theoretical abstraction and simultaneously includes evolutionary tumor developments. In an evolutionary process, tumor cells may exploit the whole extent of the rationalization features of stroma cells to implement the functional diversity of systems behavior aimed at maintaining homeostasis, and robustness in tumor systems. The introduction of genomic/non-genomic systems-directed therapeutic approaches may allow both, the uncovering of systems topologies of aggregated action effects and the broadening of therapeutic options via systems-directed approaches. Results: (1) Tumor systems biology is now turning into a scientific co-subject. (2) Developing actiontheoretical systems terms with the corresponding conceptual equipment may contribute to the classification of tumor subsystems. (3) Systems-directed therapies may meet new therapeutic requirements, which might help to create therapeutic approaches that are specifically designed for the demand of tumor stages, corresponding systems stages. Conclusions: Therefore, patients would probably not have to be selected according to age and/or co-morbidities because of known adverse toxicities of standard therapies (maximal tolerable doses). In contrast, therapies may meet the (individual) tumor systems characteristics by a systems-orientated selection of biomodulatory acting agents. As shown, toxicities may be modest.

Regression of orthotopic neuroblastoma in mice by targeting the endothelial and tumor cell compartments

BackgroundHigh-risk neuroblastoma has an overall five-year survival of less than 40%, indicating a need for new treatment strategies such as angiogenesis inhibition. Recent studies have shown that chemotherapeutic drugs can inhibit angiogenesis if administered in a continuous schedule. The aim of this study was primarily to characterize tumor spread in an orthotopic, metastatic model for aggressive, MYCN-amplified neuroblastoma and secondarily to study the effects of daily administration of the chemotherapeutic agent CHS 828 on tumor angiogenesis, tumor growth, and spread.MethodsMYCN-amplified human neuroblastoma cells (IMR-32, 2 × 106) were injected into the left adrenal gland in SCID mice through a flank incision. Nine weeks later, a new laparotomy was performed to confirm tumor establishment and to estimate tumor volume. Animals were randomized to either treatment with CHS 828 (20 mg/kg/day; p.o.) or vehicle control. Differences between groups in tumor volume were analyzed by Mann-Whitney U test and in metastatic spread using Fisher's exact test. Differences with p < 0.05 were considered statistically significant.ResultsThe orthotopic model resembled clinical neuroblastoma in respect to tumor site, growth and spread. Treatment with CHS 828 resulted in tumor regression (p < 0.001) and reduction in viable tumor fraction (p < 0.001) and metastatic spread (p < 0.05) in correlation with reduced plasma levels of the putative tumor marker chromogranin A (p < 0.001). These effects were due to increased tumor cell death and reduced angiogenesis. No treatment-related toxicities were observed.ConclusionThe metastatic animal model in this study resembled clinical neuroblastoma and is therefore clinically relevant for examining new treatment strategies for this malignancy. Our results indicate that daily scheduling of CHS 828 may be beneficial in treating patients with high-risk neuroblastoma.

Role of the Microenvironment for Radiosensitization by Patupilone

The combined treatment modality of ionizing radiation (IR) and the clinically relevant microtubule-stabilizing compound patupilone (epothilone B, EPO906) is a promising approach for anticancer therapy. Here, we investigated the role of the tumor microenvironment for the supra-additive in vivo response in tumor xenografts derived from patupilone-sensitive and patupilone-resistant non-small cell lung cancer cells. The treatment response to a combined regimen of patupilone and IR was investigated in vitro and in tumor xenografts derived from wild-type A549 and A549.EpoB40 cells, which are resistant to patupilone due to a beta-tubulin mutation. In both A549 and A549.EpoB40 cells, proliferative activity and clonogenicity were reduced in response to IR, whereas patupilone, as expected, inhibited proliferation of the mutant cell line with reduced potency. Combined treatment with patupilone and IR induced a cytotoxic effect in vitro in an additive way in A549 cells but not in the tubulin-mutated, patupilone-resistant A549.EpoB40 cells. A supra-additive tumor growth delay was induced by combined treatment in xenografts derived from A549 cells but not in xenografts derived from A549.EpoB40 cells. Histologic analysis revealed a significant decrease in tumor cell proliferation (Ki-67) and microvessel density and a treatment-dependent change of tumor hypoxia in A549 but not A549.EpoB40 xenografts. Using a genetically defined patupilone-sensitive and patupilone-resistant tumor model, we here showed that the major cytotoxic effect of the combined treatment modality of IR and patupilone is directed against the tumor cell compartment. The induced antiangiogenic effect derives indirectly from the tumor cell.

Compartment-Specific Bioluminescence Imaging (CS-BLI) High- Throughput Assays Provide Comparative Insights into the Impact of Osteoclasts Vs. Stromal Cells on Activity of Anti-Myeloma Therapeutics

Introduction: Osteoclasts (OCLs) have a central role in the pathophysiology of multiple myeloma (MM), because their excessive and unopposed activation leads to the characteristic bone lesions of this disease. However, unlike the well documented role of bone marrow stromal cells (BMSCs) in conferring resistance to diverse conventional anti- MM agents, the impact of OCLs in drug responsiveness of MM cells has not been studied extensively. This is due in part to limitations of conventional assays which are either not amenable to co-culture studies or not conducive to comprehensive high-throughput studies.Methods/Results: To address this void in the MM field, we applied compartment specific bioluminescence imaging (CS-BLI) in vitro assay in the context of MM-osteoclast co-cultures. Specifically, in these co-cultures the MM cell compartment is engineered to stably express luciferase (Luc) and is co-cultured with Luc-negative primary mature osteoclasts generated after ex vivo differentiation of peripheral blood monocytes with MCSF and RANKL. In these co-cultures, the activity of luciferase in response to addition of substrate results in bioluminescence signal which is directly proportional to number of viable Luc+ cells only, thus allowing for selective and sensitive quantification of the viable MM tumor cell compartment. By applying the CS-BLI platform in the context of MM-OCL co-cultures and comparing them with results of MM-BMSC co-cultures, we observed that mature OCLs stimulate increase in the number of viable MM cells from stroma-responsive, but not in stroma-unresponsive, cell lines. Furthermore, stroma/OCL-responsive MM cell lines that are sensitive to glucocorticoids and anthracyclines (e.g. MM-1S) exhibit significant decrease in their response to these conventional anti-MM agents when co-cultured with OCLs, similar to the resistance conferred to these agents by BMSCs. In addition, novel anti-MM agents, such as the proteasome inhibitor bortezomib and hsp90 inhibitors, were equally active against MM cells in the presence vs. absence of OCLs at concentrations and durations of drug treatment that did not significantly affect the viability of OCLs, consistent with results obtained with similar co-cultures of MM cells with BMSCs. These results confirm that BMSCs are not the only cellular compartment of the BM microenvironment that can function to support MM cell proliferation, survival and drug resistance, and that OCLs can also function in that accessory role as well.Conclusions: CS-BLI addresses key limitations that have precluded the highthroughput testing of novel agents in tumor-OCL co-cultures. Its comparative application in co-cultures of MM cells with OCL vs. BMSCs provides an opportunity to compare qualitatively and quantitatively the similarities or differences in patterns of drug resistance conferred by these accessory cell compartments. It also offers a powerful tool to identify new and, hopefully, more effective classes of drugs which are active in MM despite the effects of the BM microenvironment.

Compartment-Specific Bioluminescence Imaging (CS-BLI): A High-Throughput Approach To Identify Novel Anti-Neoplastic Therapies That Overcome the Protection of Stromal Cells.

Introduction: The bone marrow (BM) microenvironment (including bone marrow stromal cells; BMSCs) attenuates multiple myeloma (MM) cell response to conventional treatments. Novel agents (e.g. thalidomide derivatives and proteasome inhibitors) are able to overcome this BM-derived protection and consecquently have better clinical activity in relapsed/refractory MM. MM-BMSCs interactions are therefore important for testing potential anti-MM compounds. However, conventional assays used in high-throughput drug screening (e.g MTT assays) are typically not amenable to co-cultures. Conversely, assays often applied in MM-stromal co-cultures (e.g. 3H-thymidine incorporation or flow cytometry) have limitations that preclude their high-throughput application.Materials/Methods/Results: To address this void in anti-MM drug development, we established a compartment-specific bioluminescence imaging (CS-BLI) assay, where the tumor cell compartment (e.g. MM cells) is engineered to stably express luciferase (Luc) and can be co-cultured with Luc-negative accessory cells of the tumor milieu (e.g. BMSCs). The cell activity of luciferase in response to the addition of luciferin substrate in the culture results in bioluminescence signal directly proportional to number of viable Luc+ cells, thus allowing for selective and sensitive quantification of the viable MM tumor cell compartment. We established that CS-BLI exhibits linear correlation (R2>0.99) between bioluminescent signal and provides sensitive detection of viable MM cells, both in the presence and absence of BMSCs. We show that leukemia, lymphoma and solid tumor cells can also be adapted in this co-culture system, as well as different BMSC types or other accessory cells, such as fibroblasts, hepatocytes, etc can be utilized. Using the CS-BLI platform, we confirmed that Dex, alkylators and anthracyclines are less active in the presence of BMSCs, but bortezomib and hsp90 inhibitors are equally active in the presence or absence of BMSCs. We also observed that the phenomenon of stroma-mediated chemoresistance applies to not only MM cells, but also leukemic cells and solid tumor cells, e.g. breast Ca and melanoma cells. By testing a library of 80 kinase inhibitors against different MM and leukemia cells, we confirmed that CS-BLI can be applied to high-throughput drug screening of large compound libraries and be used to identify compounds the activity of which can be drastically altered by the presence of stromal cells. The majority of positive hits in our screening in the absence of BMSCs were less active when leukemic or MM cells were co-cultured with BMSCs. We also identified specific compounds that were more active in the presence of BMSCs: these latter agents would have been difficult to identify with conventional technologies.Conclusions: CS-BLI is able to overcome the key limitations that have precluded the establishment of high-throughput screening for testing new drugs in tumor-stroma co-cultures. Its application provides a powerful tool to identify new and, hopefully, more effective classes of drugs which are active despite the effects of the microenvironment.

Augmented antitumor effects of combination therapy with VEGF antibody and cisplatin on murine B16F10 melanoma cells

Our previous studies with EAC tumor model demonstrated that a VEGF polyclonal antibody combined with cisplatin inhibited tumor growth. Here we report the antitumor effect of VEGF antibody plus cisplatin on a murine metastatic tumor model specially emphasizing its effect on different angiogenic parameters both in vitro and in vivo. Mouse B16F10 melanoma cells were cultured in vitro in DMEM media containing 10% FBS, nonessential amino acids and antibiotics in a 5% CO2 incubator at 37 °C and the effect of VEGF antibody singly and in combination with cisplatin on this cell was assessed by MTT assay, matrigel invasion study and MMP-9 expression study in vitro. In vivo studies were performed by two tumor models viz B16F10 solid tumor model and B16LuF10 lung tumor model. The mice treated with VEGF antibody (PAb) alone, cisplatin alone and combination of VEGF antibody and cisplatin on alternative days from the next day of tumor transplantation. Antitumor as well as antiangiogenic efficacy was monitored by measuring tumor burden, survivability, MVD measurement, serum NO value measurement and bcl-2 expression study. It was observed that administration of combined therapy with VEGF antibody and cisplatin augmented antitumor activity in B16F10 melanoma models than the either agents alone. Thus our experiments show a successful VEGF antibody based combination therapy with cisplatin and suggests that the enhancement of antitumor activity could be explained by a concomitant effect on both endothelial and tumor cell compartment.

Inhibition of Angiogenesis and Tumor Metastasis by Targeting a Matrix Immobilized Cryptic Extracellular Matrix Epitope in Laminin

Angiogenesis and tumor metastasis depend on extracellular matrix (ECM) remodeling and subsequent cellular interactions with these modified proteins. An in-depth understanding of how both endothelial and tumor cells use matrix-immobilized cryptic ECM epitopes to regulate invasive cell behavior may lead to the development of novel strategies for the treatment of human tumors. However, little is known concerning the existence and the functional significance of cryptic laminin epitopes in regulating angiogenesis and tumor cell metastasis. Here, we report the isolation and characterization of a synthetic peptide that binds to a cryptic epitope in laminin. The STQ peptide selectively bound denatured and proteolyzed laminin but showed little interaction with native laminin. The cryptic laminin epitope recognized by this peptide was selectively exposed within malignant melanoma in vivo, whereas little if any was detected in normal mouse skin. Moreover, the STQ peptide selectively inhibited endothelial and tumor cell adhesion, migration, and proliferation in vitro and inhibited angiogenesis, tumor growth, and experimental metastasis in vivo. This inhibitory activity was associated with a selective up-regulation of the cyclin-dependent kinase inhibitor P27(KIP1) and induction of cellular senescence. These novel findings suggest the existence of functionally relevant cryptic laminin epitopes in vivo and that selective targeting of these laminin epitopes may represent an effective new strategy for the treatment of malignant tumors by affecting both the endothelial and tumor cell compartments.

Targeting tumor stroma and exploiting mature tumor vasculature to improve anti-cancer drug delivery

The identification of a critical role of tumour stroma in the regulation of tumour interstitial fluid pressure and the simultaneous discovery of the impact of anti-angiogenic drugs on tumour hemodynamics have provided new potential for improving tumour delivery of anti-cancer drugs. Here, we review the most recent studies investigating how tumour-associated fibroblasts and macrophages as well as the extracellular matrix itself may be targeted to facilitate delivery of both low-molecular weight drugs and macromolecules. In addition, we summarize the current understanding of the use of vasoactive compounds, radiotherapy and vascular-disrupting agents as potential adjuvants to maximize tumour delivery of anti-cancer drugs. The impact of these strategies on the diffusive and convective modes of drug transport is discussed in the light of Fick's and Starling's laws. Finally, we discuss how transcytosis through caveolae may also be exploited to optimize the selective delivery of conventional chemotherapy to the subendothelial tumour cell compartment.