Inflammation with restricted lysosomal proteolysis during early ascites carcinoma invasion of mouse parietal peritoneum. A medium and high-voltage electron microscopic and cytochemical study

A carcinoma invasion system (Krebs-2 and Ehrlich tetraploid ascites tumors invading mouse peritoneum) was studied by high-voltage electron microscope (HVEM) stereoscopy, conventional (medium voltage) electron microscopy (MVEM), and cytochemistry. Tumor cells entered areas of peritoneum (mainly parietal) only where mesothelial cells were damaged and where there was inflammation of the underlying stroma. The initial invasion was different from that of most other invading carcinomas in that there was minimal breakdown of basal lamina and collagen. Neither tumor cells, inflammatory leukocytes nor peritoneal fibroblasts showed significant secondary lysosome production or release of intracellular or extracellular acid phosphatase. Morphological and cytochemical criteria suggest that in some invading carcinomas, as with non-tumor migrating cells such as leukocytes, widespread proteolysis due to diffusion of proteases is not a prerequisite for invasion of stromal connective tissue.     

EGF promotes invasion by PANC-1 cells through Rac1/ROS-dependent secretion and activation of MMP-2

Cancer metastasis involves tumor cells invading the surrounding tissue. Remodeling of tissue barriers depends on the ability of tumor cells to degrade the surrounding collagen matrix and then migrate through the matrix defects. Epidermal growth factor (EGF) has been shown to regulate tumor cell invasion through activation of matrix metalloproteinase-2 (MMP-2) in various tumor cell types. In the present study, we investigated the role of MMP-2 and the signaling pathway involved in EGF-promoted invasion by human pancreatic cancer cells PANC-1. Using specific inhibitors, we found that EGF stimulation of these tumor cells induced secretion and activation of the collagenase MMP-2, which was required for EGF-stimulated basement membrane degradation and cell invasion. Our results also indicate that signaling events downstream of EGF receptor involved PI3K- and Src-dependent activation of Rac1, which mediated the NADPH-generated reactive oxygen species responsible for MMP-2 secretion and activation.     

Plasmodium sporozoite interactions with macrophages in vitro: a videomicroscopic analysis

Studies of in vitro interactions between Plasmodium berghei sporozoites and peritoneal macrophages from mice and rats were performed. A videomicroscopic analysis was made of interactions observed by phase-contrast microscopy. Our results showed a diversity of dynamic interactions between sporozoites and macrophages that included no interaction, surface interaction without sporozoite interiorization, active sporozoite penetration, active penetration with subsequent sporozoite escape, macrophage destruction, and the formation of "tethers" or web-like structures by sporozoites that had actively invaded macrophages. Sporozoites are thus clearly capable of actively invading host macrophages and are not restricted to being phagocytosed for interiorization. The formation of "tethers" by the moving sporozoite might function in vivo by anchoring the sporozoite to the cells lining the lumen of the liver sinusoid. Active sporozoite motility appears to be a functional phenomenon involved in sporozoite invasion of host liver cells.     

Mapping the immunosuppressive environment in uterine tumors: implications for immunotherapy

The major hurdle for cancer vaccines to be effective is posed by tumor immune evasion. Several common immune mechanisms and mediators are exploited by tumors to avoid immune destruction. In an attempt to shed more light on the immunosuppressive environment in uterine tumors, we analyzed the presence of PD-L1, PD-L2, B7-H4, indoleamine 2,3-dioxygenase (IDO), galectin-1, galectin-3, arginase-1 activity and myeloid-derived suppressor cell (MDSC) infiltration. IDO, PD-L1, PD-L2 and B7-H4 were analyzed by immunohistochemistry. PD-L2 was mostly expressed at low levels in these tumors. We found high IDO expression in 21 % of endometrial carcinoma samples and in 14 % of uterine sarcoma samples. For PD-L1 and B7-H4, we found high expression in 92 and 90 % of endometrial cancers, respectively, and in 100 and 92 % of the sarcomas. Galectin-1 and 3 were analyzed in tissue lysates by ELISA, but we did not find an increase in both molecules in tumor lysates compared with benign tissues. We detected expression of galectin-3 by fibroblasts, immune cells and tumor cells in single-cell tumor suspensions. In addition, we noted a highly significant increase in arginase-1 activity in endometrial carcinomas compared with normal endometria, which was not the case for uterine sarcomas. Finally, we could demonstrate MDSC infiltration in fresh tumor suspensions from uterine tumors. These results indicate that the PD-1/PD-L1 interaction and B7-H4 could be possible targets for immune intervention in uterine cancer patients as well as mediation of MDSC function. These observations are another step toward the implementation of inhibitors of immunosuppression in the treatment of uterine cancer patients.     

Collagenase inhibitors retarding invasion of a human tumor in nude mice

Tumor invasion has been correlated with the ability of tumor cells to produce collagenolytic enzymes which are capable of degrading normal host tissues. However, the human small cell carcinoma implanted subcutanouesly and growing progressively in athymic (nude) mice produced large quantities of collagenase but did not appear to significantly infultrate adjacent host tissue. In comparison, subcutaneously implanted murine Lewis lung tumors produced similar quantities of collagenase and were locally invasive. The human tumors were surrounded by a compact layer of fibroblast cells in a fibrous matrix. This fibrous sheath exhibited anticollagenase activity and indicated a mechanism of host tissue resistance to invasion via the formation of inhibitors to degradative enzymes produced by tumor cells.     

A novel host/tumor cell interaction activates matrix metalloproteinase 1 and mediates invasion through type I collagen.

Along with degradation of type IV collagen in basement membrane, destruction of the stromal collagens, types I and III, is an essential step in the invasive/metastatic behavior of tumor cells, and it is mediated, at least in part, by interstitial collagenase 1 (matrix metalloproteinase 1 (MMP-1)). Because A2058 melanoma cells produce substantial quantities of MMP-1, we used these cells as models for studying invasion of type I collagen. With a sensitive and quantitative in vitro invasion assay, we monitored the ability of these cells to invade a matrix of type I collagen and the ability of a serine proteinase inhibitor and all-trans-retinoic acid to block invasion. Although these cells produce copious amounts of MMP-1, they do not invade collagen unless they are co-cultured with fibroblasts or with conditioned medium derived from fibroblasts. Our studies indicate that a proteolytic cascade that depends on stromal/tumor cell interactions facilitates the ability of A2058 melanoma cells to invade a matrix of type I collagen. This cascade activates latent MMP-1 and involves both serine proteinases and MMPs, particularly stromelysin 1 (MMP-3). All-trans-retinoic acid (10(-6) M) suppresses the invasion of tumor cells by several mechanisms that include suppression of MMP synthesis and an increase in levels of tissue inhibitor of metalloproteinases 1 and 2. We conclude that invasion of stromal collagen by A2058 melanoma cells is mediated by a novel host/tumor cell interaction in which a proteolytic cascade culminates in the activation of pro-MMP-1 and tumor cell invasion.     

Laminin-5 gamma 2 chain expression correlates with unfavorable prognosis in colon carcinomas.

Expression of the gamma 2 chain at the invasive front of different tumors has indicated an important role for laminin-5 in cell migration during tumor invasion and tissue remodeling. As there is considerable need for reliable invasion and prognostic markers we evaluated the correlation of laminin-5 gamma 2 chain expression with clinicopathologic parameters and patient survival in 93 primary colon carcinomas. Epithelial cells of normal mucosa were consistently negative for staining. In contrast, positive cytoplasmic staining was observed in 89 tumors (96%). Twenty-four (26%) cases were scored as sparse, 34 (37%) as moderate, and 31 (33%) as frequent gamma 2 chain expression. There was a significant association of laminin-5 gamma 2 chain expression and local invasiveness of colon carcinomas according to Dukes stage (A-C) (p=0.001) and tumor budding (p<0.001). A statistical significance could also be noted in decreasing tumor differentiation (p<0.001) and correlation to tumor size (p=0.032). No correlation was observed to tumor site. Univariate analysis identified laminin-5 (p=0.010), tumor differentiation (p=0.006) and Dukes grade (p<0.001) as significant variables in predicting prognosis. However, by multivariate analyses, this study could not demonstrate that laminin-5 gamma 2 chain expression is an independent predictive factor for survival.The results indicate that laminin-5 gamma 2 chain expression is up-regulated during the progression of human colon cancer and that it plays a role in the aggressiveness of these tumors. Demonstration of laminin-5 gamma 2 chain positivity also facilitates detection of individual cells or minor cell clusters invading the surrounding stroma.Figures on http://www.esacp.org/acp/2001/22-4/lenander.htm.     

Dying and regeneration of human tumor cells after heterotransplantation to athymic mice

The histologic phenomena occurring immediately after heterotransplantation of two human colon adenocarcinomas to athymic mice have been studied. The tumors differed with respect to velocity of growth and passage age. Three phases were discernible in both cases. (1) During the first phase, most inoculated tumor cells died. (2) The second phase was characterized by removal of the necrotic tumor cells by immigrated inflammatory cells and by penetration of the connective tissue of host animals from peripheral into central areas of the implants. The first mitoses occurred within tumor cells in close proximity to these connective tissue septa. (3) During the third phase, signs of regeneration and proliferation of tumor cells resulted in the macroscopic enlargement of xenografts. Only in this phase, the typical histologic characteristics of the tumors were formed. These observations point to the host connective tissue invading into implants to be of great importance for the stimulation of tumor cell proliferation and, therefore, for the growth of xenografts. Thus, successful heterotransplantation is obviously based on mutual events between the transplanted tumor cells and host connective tissue.     

Tumor proliferation and diffusion on percolation clusters

We study in silico the influence of host tissue inhomogeneity on tumor cell proliferation and diffusion by simulating the mobility of a tumor on percolation clusters with different homogeneities of surrounding tissues. The proliferation and diffusion of a tumor in an inhomogeneous tissue could be characterized in the framework of the percolation theory, which displays similar thresholds (0.54, 0.44, and 0.37, respectively) for tumor proliferation and diffusion in three kinds of lattices with 4, 6, and 8 connecting near neighbors. Our study reveals the existence of a critical transition concerning the survival and diffusion of tumor cells with leaping metastatic diffusion movement in the host tissues. Tumor cells usually flow in the direction of greater pressure variation during their diffusing and infiltrating to a further location in the host tissue. Some specific sites suitable for tumor invasion were observed on the percolation cluster and around these specific sites a tumor can develop into scattered tumors linked by some advantage tunnels that facilitate tumor invasion. We also investigate the manner that tissue inhomogeneity surrounding a tumor may influence the velocity of tumor diffusion and invasion. Our simulation suggested that invasion of a tumor is controlled by the homogeneity of the tumor microenvironment, which is basically consistent with the experimental report by Riching et al. as well as our clinical observation of medical imaging. Both simulation and clinical observation proved that tumor diffusion and invasion into the surrounding host tissue is positively correlated with the homogeneity of the tissue.     

Studies on intercellular invasion in vitro using rabbit peritoneal neutrophil granulocytes. II. Adhesive interaction between cells.

A possible mechanism for intercellular invasion is that the strength of adhesion between host and invading cells is greater than the average of the strengths of homotypic adhesions. This hypothesis has been examined by a study of the kinetics of aggregation of dispersed populations of an invasive cell type (the rabbit peritoneal neutrophil granulocyte) and a host cell type (the chick embryo heart fibroblast) in shaken suspension culture. Since aggregation in mixed populations of the 2 cell types demonstrated tissue specificity, the hypothesis is not supported by these studies, heterotypic adhesions seem in fact to be weaker than homotypic adhesions.     

Mathematical modelling of the influence of heat shock proteins on cancer invasion of tissue

Tumour cell invasion is crucial for cancer metastasis, which is the main cause of cancer mortality. An important group of proteins involved in cancer invasion are the Heat Shock Proteins (HSPs). According to experimental data, inhibition of one of these proteins, Hsp90, slows down cancer cells while they are invading tissue, but does not affect the synthesis of matrix metalloproteinases (MMP2 and MMP9), which are very important for cancer metastasis, acting as extracellular matrix (ECM) degrading enzymes. To test different biological hypotheses regarding how precisely Hsp90 influences tumour invasion, in this paper we use a model of solid tumour growth which accounts for the interactions between Hsp90 dynamics and the migration of cancer cells and, alternatively, between Hsp90 dynamics and the synthesis of matrix degrading enzymes (MDEs). The model consists of a system of reaction-diffusion-taxis partial differential equations describing interactions between cancer cells, MDE, and the host tissue (ECM). Using numerical simulations we investigate the effects of the administration of Hsp90 inhibitors on the dynamics of tumour invasion. Alternative mechanisms of reduction of cancer invasiveness result in different simulated patterns of the invading tumour cells. Therefore, predictions of the model suggest experiments which might be performed to develop a deeper understanding of the tumour invasion process.     

Campylobacter jejuni: a study on environmental conditions affecting culturability and in vitro adhesion/invasion

Aims:  Nongrowing cultures of Campylobacter jejuni lose their culturability. It is unclear whether this loss in culturability also affects their ability to interact with host cells. The purpose of this study was to determine the relevance of the number of culturable cells to the ability to adhere/invade in Caco-2 cells.
Methods and Results:  For C. jejuni C356, culturability and adhesion/invasion were monitored in time (days) under different storage conditions (temperature, medium, atmosphere). Decrease rates of both culturability and adhesion/invasion were dependent on the conditions used, but the number of adhering/invading cells per culturable cell was not affected by the environmental conditions. Furthermore five strains were monitored at one condition. The culturability and adhesion/invasion decrease rates did not significantly differ per strain; however the number of adhering/invading cells per culturable cell was strain dependent.
Conclusions:  Culturability and adhesion/invasion of C. jejuni are linearly related. The number of adhering/invading cells per culturable C. jejuni cell is strain dependent, but is not affected by environmental conditions.
Significance and Impact of the Study:  It was shown that the number of culturable cells is a good measure for the in vitro adhesion/invasion of. C. jejuni.     

Fibroblasts-dependent invasion of podoplanin-positive cancer stem cells in squamous cell carcinoma

To clear whether podoplanin-positive cancer stem cells in squamous cell carcinoma have higher invasion activity during a fibroblasts-dependent invasion. A collagen gel invasion assay was performed using fluorescent ubiquitination-based cell cycle indicator-labeled A431 cells. The total number and number of invading cells in S/G2/M phase were counted using time-lapse imaging cocultured with fibroblasts. There was no significant difference between the number of invading podoplanin-positive and negative A431 cells when fibroblasts did not exist. On the contrary, the number of invading podoplanin-positive cells was significantly higher when fibroblasts existed. The frequency of cells in S/G2/M phase among invasion was no difference. Knockdown of podoplanin decreased the number of invaded A431 cells significantly when fibroblasts existed. Podoplanin-positive A431 cells display higher invasion activity when fibroblasts exist, suggesting that some biological functions of cancer stem cells might become evident only within the fibrous tumor microenvironment.     

Evaluation of polysialic acid in the diagnosis of Wilms' tumor. A comparative study on urinary tract tumors and non-neuroendocrine tumors

The polysialic acid moiety of the neural cell adhesion molecule has been shown to represent an onco-developmental antigen which can be detected in both embryonic human kidney and Wilms' tumor but not in normal adult human kidney. In the present comparative study, Wilms' tumors, clear cell (bone-metastasizing) sarcomas of kidney, cystic nephromas, renal cell carcinomas, transitional cell carcinomas and papillomas of the renal pelvis, ureter and urinary bladder (as well normal transitional epithelium from these regions). Ewing sarcomas, hepatoblastomas, rhabdomyosarcomas, and carcinomas of the stomach, colon, exocrine pancreas, lung, and esophagus, were investigated immunohistochemically for the presence of polysialic acid. In addition, immunoblot analysis was performed in selected tumors. With the exception of Wilms' tumor, none of the tumors investigated was positive for polysialic acid. In Wilms' tumor, blastemal cells and all epithelial components were positive but no immunostaining was observed in the stroma. These observations emphasize the potential value of a monoclonal anti-polysialic acid antibody in identifying blastemal metanephric cells and their epithelial differentiatives in Wilms' tumor.     

Mechanische Experimente an lebenden Zellen. Perspektiven für die Krebsforschung

The process of cell migration is definied by biochemical and biophysical properties of the invading cells. Accelarated investigations in the field of biomechanics are of high impact regarding the process of the migration into a 3‐D matrix of tumor cells. In contrast to use of several protein markers that determine tumor malignancy, a single cell mechanical parameter may delevop as a reliable, standard tool for tumor diagnostics. The cell mechanics of living cells shed light on tumor disease especially on the process of metastasis that involves the invasion of tumor cells through connective tissue.     

The Anomalous Diffusion of a Tumor Invading with Different Surrounding Tissues

We simulated the invasion of a proliferating, diffusing tumor within different surrounding tissue conditions using a hybrid mathematical model. The in silico invasion of a tumor was addressed systematically for the first time within the framework of a generalized diffusion theory. Our results reveal that a tumor not only migrates using typical Fickian diffusion, but also migrates more generally using subdiffusion, superdiffusion, and even ballistic diffusion, with increasing mobility of the tumor cell when haptotaxis and chemotaxis toward the host tissue surrounding the proliferative tumor are involved. Five functional terms were included in the hybrid model and their effects on a tumor''s invasion were investigated quantitatively: haptotaxis toward the extracellular matrix tissue that is degraded by matrix metalloproteinases; chemotaxis toward nutrients; cell-cell adhesion; the proliferation of the tumor; and the immune response toward the tumor. Haptotaxis and chemotaxis, which are initiated by extracellular matrix and nutrient supply (i.e., glucose) respectively, as well as cell-cell adhesions all drastically affect a tumor''s diffusion mode when a tumor invades its surrounding host tissue and proliferates. We verified the in silico invasive behavior of a tumor by analyzing experimental data gathered from the in vitro culturing of different tumor cells and clinical imaging observations that used the same approach as was used to process the simulation data. The different migration modes of a tumor suggested by the simulations generally conform to the results observed in cell cultures and in clinical imaging. Our study not only discloses some migration modes of a tumor that proliferates and invades under different host tissues conditions, but also provides a heuristic method to characterize the invasion of a tumor in clinical medical imaging analysis.     

Tight junctions adjacent to tumor stromal interface in human invasive transitional cell carcinomas.

Tight junctions adjacent to the tumor stromal interface in invading neoplastic cells of human urinary bladder carcinomas were observed. Basal lamina, collagen and elastic fibers, and cellular debris were found next to the tight junctions. An association between microenvironment (i.e., tumor necrosis) of the invading neoplastic cells and tight junction locations was suggested.     

Tumor-stroma interactions directing phenotype and progression of epithelial skin tumor cells

Tumor-stroma interactions play a significant role in tumor development and progression. Alterations in the stromal microenvironment, including enhanced vasculature (angiogenesis), modified extracellular matrix composition, inflammatory cells, and dys-balanced protease activity, are essential regulatory factors of tumor growth and invasion. Differential modulation of stromal characteristics is induced by epithelial skin tumor cells depending on their transformation stage when grown as surface transplants in vivo. Tumor cells can regulate the development of a "tumor-stroma" via the aberrant expression of growth factors or induction of growth factor receptors in the stromal compartment. In this context, secretion of the hematopoietic growth factors G-CSF and GM-CSF, constituitively expressed in enhanced malignant tumors, may be good candidates for induction of a tumor stroma through their effect on inflammatory cells. Upon its induction, the tumor stroma will reciprocally influence the differentiation status of tumor cells resulting in a normalization of benign tumor epithelia and the maintenance of a malignant phenotype, respectively. In the HaCaT model for squamous cell carcinoma of the skin, stromal activation and angiogenesis are transient in pre-malignant transplants, however they remain persistent in malignant transplants where progressive angiogenesis is closely correlated with tumor invasion. While continued expression of VEGF and PDGF are associated with benign tumor phenotypes, activation of VEGFR-2 is a hallmark of malignant tumors and accompanies ongoing angiogenesis and tumor invasion. As a consequence the inhibition of ongoing angiogenesis by blocking VEGFR-2 signalling resulted in dramatically impaired malignant tumor expansion and invasion. Comparably, tumor vascularization and invasion was blocked by disturbing the balance of matrix protease activity caused by a lack of PAI-1 in the stromal cells of the knockout mouse hosts. A similar inhibition of tumor vascularization was caused by TSP-1 over-expression in skin carcinoma cells, which also blocked tumor invasion and expansion. On the other hand, when granulation tissue and angiogenesis were only transiently activated as a result of stable transfection of PDGF into non-tumorigenic HaCaT cells, the target cells formed benign, but not malignant, tumors. Collectively, these data show that tumor vascularization, providing intimate association of blood vessels with tumor cells, is a prerequisite for tumor invasion. A potential mechanism for this interrelationship may be the differential regulation of MMP-expression in tumors of different grades of malignancy. In vitro MMP expression did not discriminate between benign and malignant tumor cells unless they were co-cultured with stromal fibroblasts. However, in vivo regulation of MMP expression was clearly dependent on tumor phenotype. While MMP-1 and MMP-13 were down-regulated in benign transplants, they were persistently up-regulated in malignant ones. A tight balance between proteases and their inhibitors is crucial for both the formation and infiltration of blood vessels and for tumor cell invasion, thus again emphasizing the importance of the stromal compartment for the development and progression of carcinomas.     

Development of the basal lamina in xenografted human carcinomas: an ultrastructural and immunohistochemical study

Summary The development of the basal lamina (BL), the key structure of the basement membrane (BM), was investigated in three xenografted human carcinomas of the sigmoid colon (CA 1), the lung (L 261), and the hypopharynx (H-Stg 1) following heterotransplantation to athymic mice. The study involved the use of electron microscopy and indirect immunofluorescence techniques employing highly specific antibodies against the intrinsic BL components, heparan sulfate proteoglycan, laminin and type-IV collagen. Following transplantation, the extracellular matrix material of the transplanted tumors decomposed and was phagocytozed by invading macrophages within 1 to 2 days. During this stage, no specific binding of the applied antibodies to BL components could be detected within the xenografts. Following the ingrowth of host-derived connective tissue between days 2 to 6, small fluorescence-positive granules appeared within the cytoplasm and around those tumor cells that were located close to the invaded strands of connective tissue. Ultrastructurally, typical secretory granules were detectable in the cytoplasm of many xenografted carcinoma cells. Thereafter, a tannic acid-positive, patchy material appeared in the extracellular space of CA 1 and L 261 and aggregated to form small fragments of a discontinuous BL. In the H-Stg 1 xenografts, this material assembled to form continuous mono-, bi- and multilayered structures. Large amounts of excess BL material remained accumulated in the L 261 and H-Stg 1 xenografts until the end of the observation period (day 24). These findings reveal that discontinuities of the BL occur independent of the active invasion processes of tumor cells, since xenografted human carcinomas neither grow invasively nor metastasize in nude mice. Moreover, they confirm that these discontinuities are not caused by a quantitatively insufficient production of BL material, but rather arise from qualitative imbalances of the composition of the synthesized BL material.