Tumor-host Interface Research Articles

Clinical study of mode of invasion in tongue squamous cell carcinoma

ObjectiveThe most commonly used system of classification for the evaluation of histological malignancy in oral squamous cell carcinoma is the Yamamoto–Kohama (Y–K) classification, which focuses on the shape of tumor cell cords at the tumor–host interface, an item also included in the Jakobsson and Anneroth classifications. In this clinical study, we investigated the relationship between mode of invasion as based on the Jakobsson and Y–K classifications and prognosis in patients with tongue squamous cell carcinoma treated at our department. Patients and methodsOne hundred and sixty-eight of the 193 patients with tongue squamous cell carcinoma treated in at our department over an 11-year period between 2000 and 2010 were included in this study (25 were excluded as the mode of invasion could not be determined according to the Y–K classification). ResultsAccording to the Jakobsson classification, 26, 33, 53, and 56 tumors were classified as grades 1–4, respectively. According to the Y–K classification, 26, 33, 53, 31, and 25 tumors were classified as grades 1–3, 4C, and 4D, respectively. ConclusionPatients with grade 4C/4D tumors had many poor prognostic factors, including a pN-positive rate of 73.5%, mortality, recurrence, and late metastasis rate of 44.2%. The tumors in this group exhibited both endophytic and exophytic growth patterns, and the 5-year-survival rate was significantly lower than that in patients with other grade tumors. These results suggest that grade 4C/4D is a useful prognostic factor.

Abstract C17: Identifying novel genes critical to invasion in head and neck squamous cell carcinoma

Abstract Oral cavity squamous cell carcinomas (OCSCC) have a poor patient prognosis, which is attributed to their invasive nature. Our goal was to identify specific genes that are important in OCSCC invasion, utilizing genome-wide expression data generated from HNSCC patient tumor samples of varying degrees of invasiveness. Invasion was assessed using the histological parameter pattern of invasion (POI), which describes the manner in which tumor cells infiltrate the normal tissue at the tumor-host interface. POI was used to categorize tumor resections into five types (1-5), type 5 being the most invasive with a “widely dispersed pattern of tumor infiltrate” and correlated significantly with the appearance of local recurrence and decreased overall survival of the patients after surgery. Global gene expression data was acquired from analysis of the surgical resections from 9 OCSCC patient samples collected at Montefiore Medical Center between 2001 and 2008. Tumor cell-enriched cores from formalin-fixed paraffin-embedded (FFPE) samples were used to generate global gene expression data using the Illumina® WG-DASL array to compare gene expression in four tumors that were WPOI type 5 and five tumors that were WPOI type 3. RNA extracted from flash-frozen tumor samples following surgical resection from the same patients was used to obtain global gene expression data using the Illumina® HumanHT-12 v4 Expression BeadChip. Candidate genes were assessed using in vitro invasion assays following siRNA knockdown of specific gene expression using siGENOME smart pools from Dharmacon. When both gene expression datasets were analyzed, we identified 104 genes that were overexpressed at least 1.5-fold in the WPOI type 5 tumors compared to the less invasive WPOI type 3 tumors. Fifty genes were initially tested based on functional criteria. In our initial screen, 16 of these genes showed a significant reduction in tumor cell invasion when expression was knocked down by siRNA in an OCSCC cell line (UMSCC1). These 16 genes are now being tested in other cell lines as well as in our in vivo mouse model to assess their invasive phenotype. This initial screen of global gene expression data in combination with pattern of invasion has revealed multiple novel genes which may play a critical role in OCSCC invasion and warrant further study. Citation Format: Sangeeta K. Jayakar, Olivier D. Loudig, Margaret Brandwein-Gensler, Kim S. Ryung, Michael B. Prystowsky, Jeffrey E. Segall, Thomas J. Belbin. Identifying novel genes critical to invasion in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C17.

Down-Regulation of the miRNA-200 Family at the Invasive Front of Colorectal Cancers with Degraded Basement Membrane Indicates EMT Is Involved in Cancer Progression

Cancer progression is a complex series of events thought to incorporate the reversible developmental process of epithelial-to-mesenchymal transition (EMT). In vitro, the microRNA-200 family maintains the epithelial phenotype by posttranscriptionally inhibiting the E-cadherin repressors, ZEB1 and ZEB2. Here, we used in situ hybridization and immunohistochemistry to assess expression of miR-200 and EMT biomarkers in formalin-fixed paraffin-embedded human colorectal adenocarcinomas. In addition, laser capture microdissection and quantitative real-time polymerase chain reaction were employed to quantify levels of miR-200 in the normal epithelium, tumor core, invasive front, and stroma. We find that miR-200 is downregulated at the invasive front of colorectal adenocarcinomas that have destroyed and invaded beyond the basement membrane. However, regional lymph node metastases and vascular carcinoma deposits show strong expression of miR-200, suggesting this family of miRNAs is involved in the recapitulation of the primary tumor phenotype at metastatic sites. In contrast, adenomas and adenocarcinomas with intact basement membranes showed uniform miR-200 expression from the tumor core to the tumor-host interface. Taken together, these data support the involvement of EMT and mesenchymal-to-epithelial transition (MET) in the metastasis cascade and show that miR-200 is downregulated in the initial stages of stromal invasion but is restored at metastatic sites.

Diversity of dynamics and morphologies of invasive solid tumors.

Complex tumor-host interactions can significantly affect the growth dynamics and morphologies of progressing neoplasms. The growth of a confined solid tumor induces mechanical pressure and deformation of the surrounding microenvironment, which in turn influences tumor growth. In this paper, we generalize a recently developed cellular automaton model for invasive tumor growth in heterogeneous microenvironments [Y. Jiao and S. Torquato, PLoS Comput. Biol.7, e1002314 (2011)] by incorporating the effects of pressure. Specifically, we explicitly model the pressure exerted on the growing tumor due to the deformation of the microenvironment and its effect on the local tumor-host interface instability. Both noninvasive-proliferative growth and invasive growth with individual cells that detach themselves from the primary tumor and migrate into the surrounding microenvironment are investigated. We find that while noninvasive tumors growing in "soft" homogeneous microenvironments develop almost isotropic shapes, both high pressure and host heterogeneity can strongly enhance malignant behavior, leading to finger-like protrusions of the tumor surface. Moreover, we show that individual invasive cells of an invasive tumor degrade the local extracellular matrix at the tumor-host interface, which diminishes the fingering growth of the primary tumor. The implications of our results for cancer diagnosis, prognosis and therapy are discussed.

Active roles of tumor stroma in breast cancer metastasis.

Metastasis is the major cause of death for breast cancer patients. Tumors are heterogenous cellular entities composed of cancer cells and cells of the microenvironment in which they reside. A reciprocal dynamic interaction occurs between the tumor cells and their surrounding stroma under physiological and pathological conditions. This tumor-host communication interface mediates the escape of tumor cells at the primary site, survival of circulating cancer cells in the vasculature, and growth of metastatic cancer at secondary site. Each step of the metastatic process is accompanied by recruitment of stromal cells from the microenvironment and production of unique array of growth factors and chemokines. Stromal microenvironment may play active roles in breast cancer metastasis. Elucidating the types of cells recruited and signal pathways involved in the crosstalk between tumor cells and stromal cells will help identify novel strategies for cotargeting cancer cells and tumor stromal cells to suppress metastasis and improve patient outcome.

Expression and role of fibroblast activation protein-alpha in microinvasive breast carcinoma

BackgroundDiagnosis of ductal carcinoma in situ (DCIS) in breast cancer cases is challenging for pathologist due to a variety of in situ patterns and artefacts, which could be misinterpreted as stromal invasion. Microinvasion is detected by the presence of cytologically malignant cells outside the confines of the basement membrane and myoepithelium. When malignant cells invade the stroma, there is tissue remodeling induced by perturbed stromal-epithelial interactions. Carcinoma-associated fibroblasts (CAFs) are main cells in the microenvironment of the remodeled tumor-host interface. They are characterized by the expression of the specific fibroblast activation protein-alpha (FAP-α), and differ from that of normal fibroblasts exhibiting an immunophenotype of CD34. We hypothesized that staining for FAP-α may be helpful in determining whether DCIS has microinvasion.Methods349 excised breast specimens were immunostained for smooth muscle actin SMA, CD34, FAP-α, and Calponin. Study material was divided into 5 groups: group 1: normal mammary tissues of healthy women after plastic surgery; group 2: usual ductal hyperplasia (UDH); group 3: DCIS without microinvasion on H & E stain; group 4: DCIS with microinvasion on H & E stain (DCIS-MI), and group 5: invasive ductal carcinoma (IDC). A comparative evaluation of the four immunostains was conducted.ResultsOur results demonstrated that using FAP-α and Calponin adjunctively improved the sensitivity of pathological diagnosis of DCIS-MI by 11.29%, whereas the adjunctive use of FAP-α and Calponin improved the sensitivity of pathological diagnosis of DCIS by 13.6%.ConclusionsThis study provides the first evidence that immunostaining with FAP-α and Calponin can serve as a novel marker for pathologically diagnosing whether DCIS has microinvasion.

Induction of Th1Immune responses following laser ablation in a murine model of colorectal liver metastases

BackgroundPreliminary experimental studies have suggested that the in situ destruction of tumor tissue by local laser ablation (LA) may also stimulate host immunity against cancer. We investigated local and systemic induction of immune responses after laser ablation in the setting of residual tumor.MethodsA murine colorectal cancer (CRC) liver metastasis model was used. Selected tumors of liver CRC bearing mice and livers of mice without tumor induction were treated with LA. Liver and tumor tissues from the ablation sites and from distant sites were collected at various time points following LA and changes in CD3+ T cells and Kupffer cells (F4/80 marker) infiltration and the expression of interferon gamma (IFNγ) were investigated by immunohistochemistry and ELISpot. Base line levels of CD3+ T cells and Kupffer cells were established in untreated mice.ResultsThe presence of tumor induced significant accumulation of CD3+ T cells and Kupffer cells at the tumor-host interface, within the tumor vascular lakes and increased their baseline concentration within the liver parenchyma. LA of the liver induced accumulation of CD3+ T-cells and Kupffer cells at the site of injury and systemic induction of immune responses as discerned by the presence of IFNγ secreting splenocytes. LA of liver tumors induced significant increase of CD3+ T-cells at site of injury, within normal liver parenchyma, and the tumor-host interface of both ablated and distant tumors. In contrast Kupffer cells only accumulated in ablated tumors and the liver parenchyma but not in distant tumors. IFNγ expression increased significantly in ablated tumors and showed an increasing trend in distant tumors.ConclusionLaser ablation in addition to local tumor destruction induces local and systemic Th1 type immune responses which may play a significant role in inhibiting tumor recurrence from residual micrometastases or circulating tumor cells.

Abstract 514: A novel dual color mouse model for studying the tumor microenvironment of pancreatic adenocarcinoma

Abstract We have developed a novel, dual color, immunocompetent mouse model for studying the pancreatic tumor microenvironment using fluorescence imaging. To develop this model, we used mice engineered to constitutively express a conditional tdTomato fluorescent transgene that converts to the expression of EGFP following Cre recombinase-mediated intramolecular rearrangement of the transgene. The reporter transgene is driven by a strong, ubiquitous promoter from a well-characterized genomic locus. Both tdTomato and EGFP fluorescent proteins are membrane-targeted, allowing distinction of single cells and cell processes within the microenvironmental setting. For our studies of the pancreatic microenvironment, we used PdxCre (Pdx-1-Cre) transgenic mice. PdxCre mice express Cre recombinase within the developing pancreas during embryogenesis. To generate mice in which the pancreatic parenchyma, expressing membrane-targeted green fluorescence (mG), is highlighted against the red fluorescent (mT) backlight of stromal and non-pancreatic tissues, we crossed PdxCre transgenic mice with conditional fluorescent (mTmG) indicator mice. Both lines are on a mixed C57Bl/6 × Sv129 background, backcrossed several generations onto C57Bl/6. We used PDA4964 pancreatic adenocarcinoma cells, derived from genetically engineered triple transgenic (LSL-KrasG12D/+;LSL-Trp53R172H/+; PdxCre) mutant mice, to generate orthotopic tumors in the pancreata of PdxCre+, mTmG+ dual transgenic, dual color mice. Using a dosage of 1 × 106 tumor cells per mouse, half of the mice developed tumors within 4-6 weeks. Ex vivo imaging of orthotopic tumor-bearing mice at necropsy revealed large pancreatic tumors comprised of non-fluorescent tumor cells supported and nourished by red fluorescent host stromal and vascular cells. The expanding tumor masses distorted and displaced the adjacent green fluorescent host pancreatic parenchyma. Upon microscopic examination of the tumor-host interface, cell borders of individual host pancreatic parenchymal and stromal cells were clearly outlined. Our preliminary findings indicate that this novel, dual color, immunocompetent mouse model will be valuable for studying the pancreatic tumor microenvironment using fluorescence imaging. 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 514. doi:10.1158/1538-7445.AM2011-514

Significance of Lgr5+ve Cancer Stem Cells in the Colon and Rectum

Although recent studies show that leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)(+ve) cells targeted by Wnt drive self-renewal in the skin and gastrointestinal organs, the clinicopathological significance of Lgr5(+ve) cancer stem cells (CSCs) of the colon remains to be elucidated. We studied the Wnt-targeted Lgr5 pathway in colorectal cancer (CRC). The expression of LGR5, c-MYC, p21CIP1/WAF1/CDKN1A, glutaminase (GLS), and miRs-23a and -23b (that target LGR5 and GLS) was evaluated by quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR). The Lgr5 protein was evaluated by immunohistochemistry. The clinical relevance of gene expression in terms of patient survival was also evaluated. Overexpression of LGR5 was significantly associated with expression of c-MYC, p21CIP1/WAF1/CDKN1A, and GLS (p<0.0001), and inversely associated with miR-23a/b (p<0.05). Immunohistochemical analysis indicated that Lgr5 may be embedded in benign adenomas, localized at the tumor-host interface, and detectable over a broad area in established tumors. High level of LGR5 expression was associated with poor prognosis for CRC cancer patients (disease-free survival; p<0.05). This study supports a significant role for LGR5 in the CSC hypothesis in CRC: (1) Lgr5(+ve) CSCs, presumably derived from normal stem cells in colonic crypts, proliferate, and the gene is overexpressed during CRC development; (2) LGR5 expression is associated with activation of Wnt pathway, including oncogenic c-MYC and high energy production via glutaminolysis; (3) LGR5 expression may be a poor prognostic factor for CRC patients. Further study of LGR5 should contribute to the development of CSC-based cancer therapeutics.

Abstract IA-20: EMT in tumor invasion and metastasis

Abstract Most colorectal adenocarcinomas (CRC) have mutations in the APC gene leading to overexpression of the Wnt-pathway effector β-catenin. By acting as a transcriptional activator, nuclear β-catenin is involved in two fundamental processes in embryonic development: epithelial to mesenchymal transition (EMT) and generation of stem cells. We have previously shown, that in particular tumor cells at the invasive front of CRCs accumulate nuclear β-catenin, undergo an EMT and aberrantly express EMT-associated transcriptional repressors, like ZEB1. The amount of such cells strongly correlates with clinical outcome and metastasis formation. We further showed that ZEB1 represses target genes involved in basement membrane formation and epithelial cell polarity. Moreover we report that ZEB1 directly suppresses transcription of members of the miRNA-200 family, which strongly activate epithelial differentiation in pancreatic, colorectal and breast cancer cells. Notably, the EMT-activators TGFβ2 and ZEB1 itself are predominant targets downregulated by these miRNAs. These results indicate that ZEB1 triggers a microRNA-mediated positive feedbackloop, which stabilizes EMT and promotes invasion of cancer cells. In contrast tumor cells in central tumor areas are differentiated and often show membranous, E-cadherin-bound β-catenin. Strikingly, metastases show again a differentiated phenotype and lack nuclear beta-catenin, indicating a mesenchymo-epithelial re-transition (MET) and a regulatory role of the tumor environment during malignant tumor progression. Based on the developmental functions of beta-catenin and our data we propose, that the EMT-associated tumor cells at the invasive front act as “migrating cancer stem cells” which can re-differentiate and, depending on the range of dissemination, give raise to the primary carcinoma and metastases. We suggest that both primary tumors and metastases are derived from a pool of EMT-associated “migrating cancer stem cells” at the tumor host interface. They are defined by strong nuclear beta-catenin accumulation and EMT-phenotype, which gives these epithelial tumor cells a feature driving malignant tumor progression including metastasis: the unusual combination of a migratory and stem cell phenotype. Citation Information: Cancer Res 2009;69(23 Suppl):IA-20.

Effect of cyhalothrin on Ehrlich tumor growth and macrophage activity in mice

Cyhalothrin, a pyrethroid insecticide, induces stress-like symptoms, increases c-fos immunoreactivity in the paraventricular nucleus of the hypothalamus, and decreases innate immune responses in laboratory animals. Macrophages are key elements in cellular immune responses and operate at the tumor-host interface. This study investigated the relationship among cyhalothrin effects on Ehrlich tumor growth, serum corticosterone levels and peritoneal macrophage activity in mice. Three experiments were done with 10 experimental (single gavage administration of 3.0 mg/kg cyhalothrin daily for 7 days) and 10 control (single gavage administration of 1.0 mL/kg vehicle of cyhalothrin preparation daily for 7 days) isogenic BALB/c mice in each experiment. Cyhalothrin i) increased Ehrlich ascitic tumor growth after ip administration of 5.0 x 106 tumor cells, i.e., ascitic fluid volume (control = 1.97 +/- 0.39 mL and experimental = 2.71 +/- 0.92 mL; P < 0.05), concentration of tumor cells/mL in the ascitic fluid (control = 111.95 +/- 16.73 x 106 and experimental = 144.60 +/- 33.18 x 106; P < 0.05), and total number of tumor cells in the ascitic fluid (control = 226.91 +/- 43.22 x 106 and experimental = 349.40 +/- 106.38 x 106; P < 0.05); ii) increased serum corticosterone levels (control = 200.0 +/- 48.3 ng/mL and experimental = 420.0 +/- 75.5 ng/mL; P < 0.05), and iii) decreased the intensity of macrophage phagocytosis (control = 132.3 +/- 19.7 and experimental = 116.2 +/- 4.6; P < 0.05) and oxidative burst (control = 173.7 +/- 40.8 and experimental= 99.58 +/- 41.7; P < 0.05) in vitro in the presence of Staphylococcus aureus. These data provide evidence that cyhalothrin simultaneously alters host resistance to Ehrlich tumor growth, hypothalamic-pituitary-adrenocortical (HPA) axis function, and peritoneal macrophage activity. The results are discussed in terms of data suggesting a link between stress, HPA axis activation and resistance to tumor growth.

Personalized immunotherapy: a siren myth?

10.2217/PME.09.39 © 2009 Future Medicine Ltd “...the antigenic characteristics of tumor cells can be perceived by the innate and cognate immune systems, which represent the extrinsic barriers against tumor progression, or at least a substantial component of the host–tumor equilibrium.” Antoine Tesniere1,2,3 1U848 INSERM, ‘Cancer, Apoptosis & Immunity, Villejuif, France 2Institut Gustave Roussy, Villejuif, France 3Universite Paris Sud, Villejuif, France Guido Kroemer1,2,3 1U848 INSERM, ‘Cancer, Apoptosis & Immunity, Villejuif, France 2Institut Gustave Roussy, Villejuif, France 3Universite Paris Sud, Villejuif, France Thomas Tursz Departement de medecine, Institut Gustave Roussy, Villejuif, France

Src Family Kinases Mediate Betel Quid-Induced Oral Cancer Cell Motility and Could Be a Biomarker for Early Invasion in Oral Squamous Cell Carcinoma

Betel quid (BQ)-chewing oral cancer is a prevalent disease in many countries of Southeast Asia. Yet, the precise disease mechanism remains largely unknown. Here, we show that BQ extract-induced cell motility in three oral cancer cells (Ca9-22, SAS, and SCC9) presumably involves the Src family kinases (SFKs). Besides, BQ extract can markedly induce cell migration of wild type mouse embryonic fibroblasts (MEFs) but not MEFs lacking three SFK members, namely, Src, Yes, and Fyn, indicating the requirement of SFKs for BQ-induced cell motility. Betel quid extract can also elevate cellular SFK activities because phosphorylation of tyrosine 416 at the catalytic domain is increased, which in turn promotes phosphorylation of an in vitro substrate, enolase. Furthermore, we identified that areca nut, a major component of BQ, is the key factor accounting for BQ-induced cell migration and invasion through SFKs-mediated signaling pathways. Immunohistochemistry revealed that, particularly in BQ-chewing cases, the activity of SFKs was significantly higher in tumor-adjacent mucosa than that in solid tumor areas (P < .01). These results suggest a possible role of SFKs in tumor-host interface and thus in early tumor invasion in vivo. Consistent with this is the observation that activation of SFKs is colocalized with invasive tumor fronts in oral squamous cell carcinoma. Together, we conclude that SFKs may represent a potential biomarker of invasion and therapeutic target in BQ-induced oral cancer.

Determination of Immunohistochemical Markers for Oral Squamous Cell Carcinoma Invasions

Carcinomas are characterized by invasion of malignant cells into the underlying connective tissue and migration of malignant cells. The cellular differentiation of the invasion site as well as the pattern of invasion is crucial for the tumor behavior. Both resection margins and the pattern of invasions are important predictors of local recurrence and survival in surgically treated patients. Therefore, surgical margins should be free from carcinoma or dysplasia after surgical intervention and the accepted definition of a close margin is tumor within 5 mm of the inked resection margin, but has not so clear yet. Others use less than 2 mm or 3 mm within one high power field or a variable definition related to the pattern of invasion at the tumor host interface. The aim of this study was to determine the immunohistochemical markers for identification of invasion pattern of oral squamous cell carcinoma (OSCC) and detection of epithelial cells of surgical margins. The study analyzed the immunohistochemical characteristics of 32 cases : 8 cases each of carcinoma in situ (CIS), OSCC border, OSCC early invasion and OSCC front using Ki-67, p53, CK 17, CK 13, laminin -5γ2 and type IV collagen. The correlation between the morphological pattern on invasive sites and immunohistochemical staining were studied. The results were obtained as follows :1)The average number of Ki-67 positive basal cells was 22.7% in CIS, 57.5% in OSCC border, 62.0% in OSCC early invasion and 60.2% in OSCC front, respectively. The percentage of immuno-positive cells in OSCC border, OSCC early invasion and OSCC front are significantly higher than those of CIS (p<0.05). p53 appeared in almost all tumor cells and highest in OSCC front.2)CK 17 was positive and strong in superficial cells of OSCC border, parabasal cells of OSCC early invasion and pearl nests of OSCC front, although the degree of positivity was variable. It was negative to discrete positive in CIS.3)CK 13 was positive in superficial and parabasal cells of CIS and pearl nests of OSCC front, negative in basal cells of CIS and OSCC early invasion, and weakly present in variable distribution in OSCC border.4)The cytoplasmic accumulation of laminin -5γ2 expression was intense in OSCC border, moderate in OSCC early invasion and OSCC front, and discrete in CIS.5)Type IV collagen was strong in both OSCC border and OSCC early invasion and weak in CIS and OSCC front.These results suggested that the combined expression of Ki-67 and p53 confirmed the proliferative and mutational activities of these OSCC invasions in contrast to CIS. CK 17 and CK 13 biomarkers detected their histological differentiation and variation in epithelial cells. The migration-promoting activity and role in cancer invasion were regulated by laminin -5γ2 and type IV collagen, and was clearly observed in OSCC border. The present study also revealed high proliferative activity and high invasiveness of epithelial cells in OSCC border similar to cancer and the combined used of the markers for safety margins should be considered important by surgeon.

The first Sino-German symposium on cancer research, Shanghai May 2007

This meeting report gives a short overview on the contributions of 17 leading scientists from China and Germany. The symposium was supported by the SinoGermany Science Center Bejing, the Deutsche Forschungsgemeinschaft and the National Science Foundation, China. It intended to foster communication and collaboration of researchers from both countries. The “Migrating Cancer Stem Cell”—Concept was discussed by Thomas Brabletz (Dept. of Surgery Univ. of Freiburg). Most colorectal adenocarcinomas (CRC) have mutations in the APC gene leading to overexpression of the Wnt-pathway effector beta-catenin. By acting as a transcriptional activator, nuclear beta-catenin is decisively involved in two fundamental processes in embryonic development: epithelial to mesenchymal transition (EMT) and generation of stem cells (e.g., in intestine development). We have previously shown that nuclear beta-catenin accumulates in tumor cells at the invasive front undergoing an EMT. The amount of these cells strongly correlates with clinical outcome and metastasis formation. In contrast, tumor cells in central tumor areas are differentiated and often show membranous, E-cadherin-bound beta-catenin. Strikingly, also, metastases show again a differentiated phenotype lacking nuclear beta-catenin, indicating a regulatory role of the tumor environment for malignant progression. Based on the developmental functions of beta-catenin and our data, we propose that both primary tumors and metastases are derived from the pool of EMT-associated “migrating cancer stem cells” at the tumor host interface, which are defined by strong nuclear beta-catenin accumulation and expression of its target genes. This gives these cells a feature, which drives malignant tumor progression including metastasis: The unusual combination of abilities allowing simultaneously to migrate and behave as a cancer stem cell. Molecular Targets in Cancer Therapy—New Possibilities and Old Limitations were reported by Reinhard Buettner (Inst. of Pathology, University of Bonn). Increasing knowledge on molecular pathogenesis of malignant tumors and activated signaling pathways has led to the concept of targeted therapies. Small molecularly designed molecules target specifically oncoproteins, and thereby modulate growth, apoptosis, or invasion of cancer cells. Currently, most experiences exit with membrane-coupled growth factor receptors and their intracellularly coupled signaling kinases. In cancer cells, these tyrosine kinase-triggered signaling pathways are frequently altered and causally involved in malignant transformation. Indeed, some of the tyrosine Virchows Arch (2007) 451:1099–1104 DOI 10.1007/s00428-007-0488-2

Quantification of in vivo tumor invasion and vascularization by computerized image analysis

The matrix-inserted surface transplantation model is an in vivo assay used to analyse the kinetics of tumor–vessel interactions during different stages of skin carcinoma progression. This system allows the study of host–tumor interface, i.e. penetration of tumor cells into normal host tissue as well as infiltration of normal host cells into the tumor. In the present study, image analysis algorithms for processing and quantifying the extent of such migratory and tissue remodeling events are presented. The proposed method is non-parametric and its originality lies in its particularity to take into account the specific geometry of tumor–host interface. This methodology is validated by evaluating the contribution of matrix metalloproteases (MMPs) in skin carcinoma invasion and vascularization through pharmacological and genetic approaches.

Predictive oncology: A review of multidisciplinary, multiscale in silico modeling linking phenotype, morphology and growth

Empirical evidence and theoretical studies suggest that the phenotype, i.e., cellular- and molecular-scale dynamics, including proliferation rate and adhesiveness due to microenvironmental factors and gene expression that govern tumor growth and invasiveness, also determine gross tumor-scale morphology. It has been difficult to quantify the relative effect of these links on disease progression and prognosis using conventional clinical and experimental methods and observables. As a result, successful individualized treatment of highly malignant and invasive cancers, such as glioblastoma, via surgical resection and chemotherapy cannot be offered and outcomes are generally poor. What is needed is a deterministic, quantifiable method to enable understanding of the connections between phenotype and tumor morphology. Here, we critically assess advantages and disadvantages of recent computational modeling efforts (e.g., continuum, discrete, and cellular automata models) that have pursued this understanding. Based on this assessment, we review a multiscale, i.e., from the molecular to the gross tumor scale, mathematical and computational “first-principle” approach based on mass conservation and other physical laws, such as employed in reaction-diffusion systems. Model variables describe known characteristics of tumor behavior, and parameters and functional relationships across scales are informed from in vitro, in vivo and ex vivo biology. We review the feasibility of this methodology that, once coupled to tumor imaging and tumor biopsy or cell culture data, should enable prediction of tumor growth and therapy outcome through quantification of the relation between the underlying dynamics and morphological characteristics. In particular, morphologic stability analysis of this mathematical model reveals that tumor cell patterning at the tumor–host interface is regulated by cell proliferation, adhesion and other phenotypic characteristics: histopathology information of tumor boundary can be inputted to the mathematical model and used as a phenotype-diagnostic tool to predict collective and individual tumor cell invasion of surrounding tissue. This approach further provides a means to deterministically test effects of novel and hypothetical therapy strategies on tumor behavior.

Acid-Mediated Tumor Invasion: a Multidisciplinary Study

The acid-mediated tumor invasion hypothesis proposes altered glucose metabolism and increased glucose uptake, observed in the vast majority of clinical cancers by fluorodeoxyglucose-positron emission tomography, are critical for development of the invasive phenotype. In this model, increased acid production due to altered glucose metabolism serves as a key intermediate by producing H(+) flow along concentration gradients into adjacent normal tissue. This chronic exposure of peritumoral normal tissue to an acidic microenvironment produces toxicity by: (a) normal cell death caused by the collapse of the transmembrane H(+) gradient inducing necrosis or apoptosis and (b) extracellular matrix degradation through the release of cathepsin B and other proteolytic enzymes. Tumor cells evolve resistance to acid-induced toxicity during carcinogenesis, allowing them to survive and proliferate in low pH microenvironments. This permits them to invade the damaged adjacent normal tissue despite the acid gradients. Here, we describe theoretical and empirical evidence for acid-mediated invasion. In silico simulations using mathematical models provide testable predictions concerning the morphology and cellular and extracellular dynamics at the tumor-host interface. In vivo experiments confirm the presence of peritumoral acid gradients as well as cellular toxicity and extracellular matrix degradation in the normal tissue exposed to the acidic microenvironment. The acid-mediated tumor invasion model provides a simple mechanism linking altered glucose metabolism with the ability of tumor cells to form invasive cancers.

Reduction of Experimental Human Fibrosarcoma Lung Metastasis in Mice by Adenovirus-Mediated Cystatin C Overexpression in the Host

Tumor cell invasion and metastasis are associated with degradation of components of the extracellular matrix by different proteinases. Among those, papain-like cysteine proteases, such as cathepsin B, seem to play an important role, as they are associated with poor clinical outcome in different cancers. In this study, we tested whether cystatin C, a natural extracellular inhibitor of papain-like cysteine proteases, can inhibit metastasis when overexpressed at the tumor-host interface. Local overexpression of cystatin C in liver and lungs of CD1 nu/nu mice was achieved by gene transfer with a novel adenoviral construct, which also led to the presence of 60 ng/mL of cystatin C in the serum. Three days after gene transfer, these mice were challenged by i.v. inoculation of lacZ-tagged human fibrosarcoma cells (HT1080lacZ-K15), leading to the formation of experimental lung and liver metastases. In this model, formation of experimental metastatic foci correlated with expression of cathepsin B in lungs, whereas there was no correlation with metastasis to the liver. In mice overexpressing cystatin C, the number of lung metastases was significantly reduced by 92%, as compared with mice receiving control adenovirus. The efficacy of extravasation of HT1080lacZ-K15 cells into the liver was not affected, indicating the independence of this process from the activity of cysteine-cathepsins. The present report is the first evidence of successful reduction of metastasis by inhibition of cysteine-cathepsins by cystatin C overexpression in the host microenvironment. Furthermore, organ-specific protease expression during tumor-host cell interactions could affect the success of antiproteolytic intervention against metastasis.

Labeling fibroblasts with biotin‐BSA‐GdDTPA‐FAM for tracking of tumor‐associated stroma by fluorescence and MR imaging

Fibroblasts at the tumor-host interface can differentiate into myofibroblasts and pericytes, and contribute to the guidance and stabilization of endothelial sprouts. After intravenous administration of biotin-BSA-GdDTPA-FAM in mice with subcutaneous MLS human ovarian carcinoma tumors, the distribution of the macromolecular MRI/optical contrast material was confined to blood vessels in normal tissues, while it co-registered with alphaSMA-positive stroma tracks within the tumor. These alphaSMA-positive tumor-associated myofibroblasts and pericytes showed uptake of the contrast material into intracellular granules. We evaluated the use of this contrast material for in vitro labeling of tumor fibroblasts as an approach for tracking their involvement in angiogenesis. Fluorescence microscopy demonstrated internalization of the contrast material, and MRI revealed a significant increase in the R(1) relaxation rate of labeled fibroblasts. R(1) not only remained elevated for 2 weeks in culture, it also increased with cell proliferation, indicating prolonged retention of the contrast material and subsequent intracellular processing and redistribution of the material, and thereby enhancing MR contrast. Moreover, cells that were labeled ex vivo with MR contrast material and co-inoculated with tumor cells in mice were detected in vivo by MRI. Uptake of the contrast material was suppressed by nystatin, suggesting internalization by caveolae-mediated endocytosis. This study shows that labeling of fibroblasts with biotin-BSA-GdDTPA-FAM is feasible and would allow noninvasive in vivo tracking of fibroblasts during tumor angiogenesis and vessel maturation.