Cell Budding from Normal Appearing Epithelia: A Predictor of Colorectal Cancer Metastasis?

Background: Colorectal carcinogenesis is believed to be a multi-stage process that originates with a localized adenoma, which linearly progresses to an intra-mucosal carcinoma, to an invasive lesion, and finally to metastatic cancer. This progression model is supported by tissue culture and animal model studies, but it is difficult to reconcile with several well-established observations, principally among these are that up to 25% of early stage (Stage I/II), node-negative colorectal cancer (CRC) develop distant metastasis, and that circulating CRC cells are undetectable in peripheral blood samples of up to 50% of patients with confirmed metastasis, but more than 30% of patients with no detectable metastasis exhibit such cells. The mechanism responsible for this diverse behavior is unknown, and there are no effective means to identify patients with pending, or who are at high risk for, developing metastatic CRC.Novel findings: Our previous studies of human breast and prostate cancer have shown that cancer invasion arises from the convergence of a tissue injury, the innate immune response to that injury, and the presence of tumor stem cells within tumor capsules at the site of the injury. Focal degeneration of a capsule due to age or disease attracts lymphocyte infiltration that degrades the degenerating capsules resulting in the formation of a focal disruption in the capsule, which selectively favors proliferating or “budding” of the underlying tumor stem cells. Our recent studies suggest that lymphocyte infiltration also triggers metastasis by disrupting the intercellular junctions and surface adhesion molecules within the proliferating cell buds causing their dissociation. Then, lymphocytes and tumor cells are conjoined through membrane fusion to form tumor-lymphocyte chimeras (TLCs) that allows the tumor stem cell to avail itself of the lymphocyte''s natural ability to migrate and breach cell barriers in order to intravasate and to travel to distant organs. Our most recent studies of human CRC have detected nearly identical focal capsule disruptions, lymphocyte infiltration, budding cells, and the formation of TLCs. Our studies have further shown that age- and type-matched node-positive and -negative CRC have a significantly different morphological and immunohistochemical profile and that the majority of lymphatic ducts with disseminated cells are located within the mucosa adjacent to morphologically normal appearing epithelial structures that express a stem cell-related marker.New hypothesis: Based on these findings and the growth patterns of budding cells revealed by double immunohistochemistry, we further hypothesize that metastatic spread is an early event of carcinogenesis and that budding cells overlying focal capsule disruptions represent invasion- and metastasis-initiating cells that follow one of four pathways to progress: (1) to undergo extensive in situ proliferation leading to the formation of tumor nests that subsequently invade the submucosa, (2) to migrate with associated lymphocytes functioning as “seeds” to grow in new sites, (3) to migrate and intravasate into pre-existing vascular structures by forming TLCs, or (4) to intravasate into vascular structures that are generated by the budding cells themselves. We also propose that only node-positive cases harbor stem cells with the potential for multi-lineage differentiation and unique surface markers that permit intravasation.     

Fluid movement and joint capsule strains due to flexion in rabbit knees

Diarthrodial joints are freely moveable joints containing synovial fluid (SF) within a connective tissue joint capsule that allows for low-friction and low-wear articulation of the cartilaginous ends of long bones. Biomechanical cues from joint articulation regulate synoviocyte and cartilage biology via joint capsule strain, in turn altering the composition of SF. Joint flexion is clinically associated with pain in knees with arthritis and effusion, with the nociception possibly originating from joint capsule strain. The hypothesis of this study was that knee fluid volume distribution and joint capsule strain are altered with passive flexion in the rabbit model. The aims were to (a) determine the volume distribution of fluid in the joint at different total volumes and with flexion of rabbit knees ex vivo, (b) correlate the volume distribution for the ex vivo model to in vivo data, and (c) determine the strains at different locations in the joint capsule with flexion. During knee flexion, ～20% of anteriorly located joint fluid moved posteriorly, correlating well with the fluid motion observed in in vivo joints. Planar joint capsule principal strains were ～100% (tension) in the proximal-distal direction and ～-40% (shortening) in the circumferential direction, relative to the femur axis and 30° strain state. The joint capsule strains with flexion are consistent with the mechanics of the tendons and ligaments from which the capsule tissue is derived. The movement and mixing of SF volume with flexion determine the mechanical and biological fluid environment within the knee joint. Joint fluid movement and capsular strains affect synovial cell biology and likely modulate trans-synovial transport.     

Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease-activated receptor-1

The coagulation protease thrombin plays a critical role in hemostasis and exerts pro-inflammatory and pro-fibrotic effects via proteolytic activation of the major thrombin receptor, protease-activated receptor-1 (PAR-1). Connective tissue growth factor (CTGF) is a novel fibroblast mitogen and also promotes extracellular matrix protein production. It is selectively induced by transforming growth factor beta (TGF-beta) and is thought to be the autocrine agent responsible for mediating its pro-fibrotic effects. CTGF is up-regulated during tissue repair and in fibrotic conditions associated with activation of the coagulation cascade. We therefore hypothesized that coagulation proteases promote the production of CTGF by cells at sites of tissue injury. To begin to address this hypothesis, we assessed the effect of coagulation proteases on fibroblast CTGF expression in vitro, and we show that thrombin, at physiological concentrations, up-regulated CTGF mRNA levels 5-fold relative to base line (p < 0.01) in fetal fibroblasts and 7-fold in primary adult fibroblasts (p < 0.01). These effects were cycloheximide-insensitive and were not blocked with a pan-specific TGF-beta-neutralizing antibody. They were further paralleled by a concomitant increase in CTGF protein production and could be mimicked with selective PAR-1 agonists. In addition, fibroblasts derived from PAR-1 knockout mice were unresponsive to thrombin but responded normally to TGF-beta(1). Finally, factor Xa, which is responsible for activating prothrombin during blood coagulation, exerted similar stimulatory effects. We propose that coagulation proteases and PAR-1 may play a role in promoting connective tissue formation during normal tissue repair and the development of fibrosis by up-regulating fibroblast CTGF expression.     

Collective cell migration in morphogenesis and cancer

The movement of cells that maintain cell-cell junctions yet protrude along or within tissues is an important mechanism for cell positioning in morphogenesis, tissue repair and cancer. Collective cell migration shares similarities but also important differences to individually migrating cells. Coherent groups of cells are arranged and held together by cell-cell adhesion molecules, including cadherins, integrins, ALCAM and NCAM. Integrins of the beta 1 and beta 3 families further provide polarized interactions with the extracellular tissue environment, while matrix-degrading proteases become focalized to substrate contacts to widen tissue space for the advancing cell mass. By generating one functional unit, in contrast to individual cell migration, collective migration provides the active and passive translocation of mobile and non-mobile cells, respectively. This review highlights cellular and molecular principles of collective migration in the context of morphogenic tissue patterning and tumor cell invasion.     

Expression of acute and late-stage inflammatory antigens, c-fms, CSF-1, and human monocytic serine esterase 1, in tumor-associated macrophages of renal cell carcinomas

Purpose: Tumor cells influence the differentiation of infiltrating macrophages. In the present study, the differentiation of macrophages in renal cell carcinomas was investigated with special regard to their possible role in tumor growth and spread. Methods: Macrophages were characterized by means of immunohistochemistry of the Ki-M1P, 25F9, MRP8, MRP14, and MRP8/14 antigens and by means of in situ hybridization of CSF-1, its c-fms-coded corresponding receptor, and human monocytic serine esterase-1 (HMSE-1) mRNA. Macrophage subgroups were quantified within central tumor tissue, the corresponding tumor host interface, and tumor-free tissue and correlated with tumor necrosis, fibrosis, and tumor stage and grade. Results: Macrophage density was much higher within tumor tissue and the tumor/host interface than in tumor-free tissue. Well-differentiated carcinomas showed a lower degree of macrophage density than less-differentiated carcinomas. Tumor-associated macrophages could be divided into an active inflammatory type (MRP14⁺, MRP8/14⁺) and into a late-phase inflammatory type (25F9⁺, MRP8⁺). Necrosis was seen in less-differentiated carcinomas and associated with a significantly increased density of MRP14⁺ macrophages, which, however, did not correlate with the extent of necrosis. The density of 25F9⁺ macrophages was correlated with an extensive connective tissue formation and an advanced tumor stage. c-fms, CSF-1, and HMSE-1 mRNA expression did not discriminate between the macrophage subgroups. Conclusions: Tumor-associated macrophages of the late-stage inflammatory type potentially support the spread of renal cell cancer. CSF-1 derived from tumor cells and macrophages acts as a monocyte attractant and induces macrophage differentiation able to modulate the extracellular matrix rather than to exert cytotoxicity. Received: 25 May 2000 / Accepted: 29 June 2000     

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.     

Racing against host's immunity defenses: a likely strategy for passive evasion of encapsulation in Asobara tabida parasitoids

The hymenopteran Asobara tabida Nees (Braconidae, Alysiinae) develops as a solitary endophagous parasite in larvae of several Drosophila species. Most A. tabida eggs possess a sticky chorion which attaches to the tissue of the host organs within a few hours following oviposition. A. tabida sticky eggs usually avoid encapsulation, though the probability of survival decreases in hosts carrying a larger number of circulating hemocytes. Here, we hypothesized that the elicitation of the encapsulation reaction may result from a race between two phenomena: the host's hemocytic reaction and the embedment of the parasitic egg within the host tissues. In order to test this hypothesis, we measured the speed of capsule formation in D. melanogaster larvae of different ages, knowing that the number of circulating hemocytes increases with the age of the larvae. Using a non-virulent A. tabida strain, the eggs of which do not attach to the host tissue, we found that the speed of capsule formation increased correlatively with the age of the D. melanogaster larva. Therefore, the hypothesis of a physiological race between host's immunity defenses and parasite's avoidance of host's defenses is strongly supported by our results. Also, A. tabida eggs which attach to the host's tissue before the attack by the hemocytes has taken place may be considered as a strategy of passive evasion from encapsulation.     

Target-selective activation of a TNF prodrug by urokinase-type plasminogen activator (uPA) mediated proteolytic processing at the cell surface

We have previously developed TNF prodrugs comprised of a N-terminal scFv targeting, a TNF effector and a C-terminal TNFR1-derived inhibitor module linked to TNF via a MMP-2 motif containing peptide, allowing activation by MMP-2-expressing tumor cells. To overcome the known heterogeneity of matrix metalloprotease expression, we developed TNF prodrugs that become processed by other tumor and/or stroma-associated proteases. These TNF prodrugs comprise either an uPA-selective or a dual uPA-MMP-2-specific linker which displayed efficient, target-dependent and cleavage sequence-specific activation by the corresponding tumor cell-expressed proteases. Selective pharmacologic inhibition of endogenous uPA and MMP-2 confirm independent prodrug processing by these two model proteases and indicate the functional superiority of a prodrug containing a multi-specific protease linker. Processing optimised TNF prodrugs should increase the proportion of active therapeutic within the targeted tissue and thus potentially enhance tumor response rate.Authors Jeannette Gerspach and Julia Németh have contributed equally to this work     

Mast cells in tumor growth: Angiogenesis,tissue remodelling and immune-modulation

There is a growing acceptance that tumor-infiltrating myeloid cells play an active role in tumor growth and mast cells are one of the earliest cell types to infiltrate developing tumors. Mast cells accumulate at the boundary between healthy tissues and malignancies and are often found in close association with blood vessels within the tumor microenvironment. They express many pro-angiogenic compounds, and may play an early role in angiogenesis within developing tumors. Mast cells also remodel extracellular matrix during wound healing, and this function is subverted in tumor growth, promoting tumor spread and metastasis. In addition, mast cells modulate immune responses by dampening immune rejection or directing immune cell recruitment, depending on local stimuli. In this review, we focus on key roles for mast cells in angiogenesis, tissue remodelling and immune modulation and highlight recent findings on the integral role that mast cells play in tumor growth. New findings suggest that mast cells may serve as a novel therapeutic target for cancer treatment and that inhibiting mast cell function may lead to tumor regression.     

Conjunction of tumor cells with lymphocytes: implications for tumor invasion and metastasis

Our previous studies have led to a novel hypothesis that tumor metastasis is triggered by aberrant lymphocyte infiltration that disrupts intercellular junctions and surface adhesion molecules and causes dissociation of tumor cells from the primary tumor core, allowing lymphocytes to conjoin with dissociated tumor cells and physically 'drag' them to different tissue sites. Our hypothesis is supported by morphological and immunohistochemical data from multiple types of human cancer. This hypothesis challenges the traditional belief that the physical conjunction between tumor cells and lymphocytes would lead to degeneration of the tumor cells. To validate our hypothesis, H&E and immunostained sections were examined under high magnification to identify potential signs of degeneration-related changes. Our study revealed that >60% of isolated tumor cells overlying focal capsule disruptions, or within the stroma and vascular structures, were physically conjoined with lymphocytes to form tumor cell-lymphocyte chimeras (TLCs). Approximately 90% of the tumor cell partners of TLCs were morphologically indistinguishable from their counterparts within the tumor core. In addition, one third of the tumor cells of TLCs expressed high levels of cell proliferation specific proteins, or were undergoing mitosis. Our study also revealed that a subset of dilated lymphatic ducts or blood vessels at the site of focal capsule disruptions harbored variable numbers of tumor cells, and the wall of these structures was in direct physical continuity with the myoepithelial cell layer. Our study suggests that the onset of tumor metastasis may occur in two forms: (1) lymphocyte-mediated shuttling that allows lymphocytes to physically 'drag' tumor cells to different sites, and (2) tumor progenitor-mediated angiogenesis that allows tumor cells to directly enter the vascular structures.     

Amino acid sequence of a mouse mucosal mast cell protease

The amino acid sequence has been determined of a mouse mucosal mast cell protease isolated from the small intestines of mice infected with Trichinella spiralis. The active protease contains 226 residues. Those corresponding to the catalytic triad of the active site of mammalian serine proteases (His-57, Asp-102, and Ser-195 in chymotrypsin) occur in identical positions. A computer search for homology indicates 74.3% and 74.1% sequence identity of the mouse mast cell protease compared to those of rat mast cell proteases I and II (RMCP I and II), respectively. The six half-cystine residues in the mouse mast cell protease are located in the same positions as in the rat mast cell proteases, cathepsin G, and the lymphocyte proteases, suggesting that they all have identical disulfide bond arrangements. At physiological pH, the mouse and rat mucosal mast cell proteases have net charges of +3 and +4, respectively, as compared to +18 for the protease (RMCP I) from rat connective tissue mast cells. This observation is consistent with the difference in solubility between the mucosal and connective tissue mast cell proteases when the enzymes are extracted from their granules under physiological conditions.     

VEGF-D promotes the metastatic spread of tumor cells via the lymphatics

Metastasis to local lymph nodes via the lymphatic vessels is a common step in the spread of solid tumors. To investigate the molecular mechanisms underlying the spread of cancer by the lymphatics, we examined the ability of vascular endothelial growth factor (VEGF)-D, a ligand for the lymphatic growth factor receptor VEGFR-3/Flt-4, to induce formation of lymphatics in a mouse tumor model. Staining with markers specific for lymphatic endothelium demonstrated that VEGF-D induced the formation of lymphatics within tumors. Moreover, expression of VEGF-D in tumor cells led to spread of the tumor to lymph nodes, whereas expression of VEGF, an angiogenic growth factor which activates VEGFR-2 but not VEGFR-3, did not. VEGF-D also promoted tumor angiogenesis and growth. Lymphatic spread induced by VEGF-D could be blocked with an antibody specific for VEGF-D. This study demonstrates that lymphatics can be established in solid tumors and implicates VEGF family members in determining the route of metastatic spread.     

The structure and functional significance of variations in the connective tissue within muscle

The amount of intramuscular connective tissue (IMCT) and its morphological distribution is highly variable between muscles of differing function. The functional roles of this component of muscle have been poorly understood, but a picture is gradually emerging of the central role this component has in growth, transmission of mechanical signals to muscle cells and co-ordination of forces between fibres within a muscle. The aim of this review is to highlight recent advances that begin to show the functional significance of some of the variability in IMCT. IMCT has a number of clearly defined roles. It patterns muscle development and innervation, and mechanically integrates the tissue. In developing muscles, proliferation and growth of muscle cells is stimulated and guided by cell-matrix interactions. Recent work has shown that the topography of collagen fibres is an important signal. The timing and rates of expression of connective tissue proteins also show differences between muscles. Discussion of mechanical roles for IMCT has traditionally been limited to the passive elastic response of muscle. However, it is now clear that IMCT provides a matrix to integrate the contractile function of the whole tissue. Mechanical forces are co-ordinated and passed between adjacent muscle cells via cell-matrix interactions and the endomysial connective tissue that links the cells together. An emerging concept is that division of a muscle into fascicles by the perimysial connective tissue is related to the need to accommodate shear strains as muscles change shape during contraction and extension.     

Activation of human breast carcinoma collagenase through plasminogen activator

Latent collagenase activity was detected in the media of a well-characterized line of human breast carcinoma cells maintained for over two years in culture. The media also contained sufficient plasminogen activator to convert extrinsically added plasminogen to plasmin which in turn activated the collagenase. During culture of the breast carcinoma in serum-free medium, collagenase activity was maximum on day 12 whereas plasminogen activator activity changed little with time. Using type I collagen as a substrate, the activated breast tumor collagenase produced $\text{3}\text{4}\text{?}\text{1}\text{4}$ fragments consistent with a mammalian collagenase. These findings suggest a pathologic role of plasminogen activator in the activation of latent collagenase during tumor invasion.A number of investigators have postulated that proteases may play a role in tumor invasion (1–5). Collagenase is one such protease which is active at neutral pH and specifically cleaves triple helical collagen into two ($\text{3}\text{4}\text{?}\text{1}\text{4}$ fragments (6). Secretion of collagenase by tumor cells migrating from the primary mass provides an attractive hypothesis for the mechanism of tumor invasion of surrounding host connective tissue—since the local environment would likely be at neutral pH. Consequently, a number of investigators have reported significant levels of collagenase activity in a wide variety of tumors (7–14). Abramson (13) has correlated aggressive $\text{in vivo}$ growth in carcinomas of the head and neck with collagenase activity, and Kuettner et al. (14) have postulated that inhibitors of collagenase may prevent tumors from invading cartilage.Collagenase is produced in both latent and active forms (6). The latent form can be activated with brief protease treatment (15). Since one of the proteases capable of activating collagenase is plasmin (15), the possibility arose that tumor cells could activate collagenase through plasminogen activator. Plasminogen activator secreted by tumor cells (4, 5) could convert plasminogen zymogen to plasmin which would in turn activate latent tumor collagenase. Testing this hypothesis $\text{in vitro}$ was the subject of the present study.Previous studies on collagenase from human carcinoma (7, 13, 14) have suffered from the drawback that contaminating inflammatory cells and fibroblasts may have been the source of the collagenase. Therefore, we have studied collagenase production from cultured human breast carcinoma cells which have been well characterized to be mammary epithelial in origin, malignant in karyotype, and able to grow in nude mice. Production of collagenase from these cells is therefore unequivocally of human carcinoma origin. The time course of latent collagenase and plasminogen activator secretion by these cultured tumor cells was studied following withdrawal of serum. To test whether plasminogen activator was secreted in sufficient amounts to indirectly activate latent collagenase, collagenase activity of the culture media was studied after the extrinsic addition of plasminogen. Finally, to verify that the tumor-secreted collagenase cleaved type I collagen at a single locus, enzyme degradation products were studied by gel electrophoresis.     

Contractile forces in tumor cell migration

Cancer is a deadly disease primarily because of the ability of tumor cells to spread from the primary tumor, to invade into the connective tissue, and to form metastases at distant sites. In contrast to cell migration on a planar surface where large cell tractions and contractile forces are not essential, tractions and forces are thought to be crucial for overcoming the resistance and steric hindrance of a dense three-dimensional connective tissue matrix. In this review, we describe recently developed biophysical tools, including 2-D and 3-D traction microscopy to measure contractile forces of cells. We discuss evidence indicating that tumor cell invasiveness is associated with increased contractile force generation.     

Differences in tetranectin immunoreactivity between benign and malignant breast tissue.

Tetranectin (TN) is a human, plasminogen kringle 4 binding plasma protein with ubiquitous cellular distribution and lectin-like characteristics. By means of the peroxidase-antiperoxidase staining technique a polyclonal and a monoclonal antibody were used to demonstrate TN within the intracellular as well as the extracellular compartment of invasive breast carcinoma. Whereas cell associated TN was universal showing only quantitative differences depending of the growth pattern of the tumor, 78 of 133 tumors displayed TN extracellularly as well. The occurrence of this stromal TN immunoreactivity was closely associated with desmoplasia, recognized morphologically by an increase in fibroblastic cells and immunohistochemically by an intense staining for the connective tissue glycoprotein fibronectin (FN). Benign breast tissue displayed a universal, intense cytoplasmic but no extracellular reaction for TN, with the exception of rare foci of granulation tissue and around dilated cysts. Functional studies have shown that human embryonal lung fibroblasts increase their release of TN to the growth medium upon stimulation. The presence of TN extracellularly within fibroblast-rich foci of desmoplasia (and granulation tissue) suggests that a similar increased release of the protein takes place in vivo during active states. Desmoplasia has been found to have a protective effect on tumor cell propagation and metastasis in a murine model. The molecular interactions, which are responsible for this effect, are undoubtedly complex. However, TN may, by its specific binding to kringle 4 of plasminogen and its high affinity for sulphated polysaccharides, add to the understanding of how plasminogen activation is modulated at the local extracellular level.     

Differences in tetranectin immunoreactivity between benign and malignant breast tissue

Summary Tetranectin (TN) is a human, plasminogen kringle 4 binding plasma protein with ubiquitous cellular distribution and lectin-like characteristics. By means of the peroxidase-antiperoxidase staining technique a polyclonal and a monoclonal antibody were used to demonstrate TN within the intracellular as well as the extracellular compartment of invasive breast carcinoma. Whereas cell associated TN was universal showing only quantitative differences depending of the growth pattern of the tumor, 78 of 133 tumors displayed TN extracellularly as well. The occurrence of this stromal TN immunoreactivity was closely associated with desmoplasia, recognized morphologically by an increase in fibroblastic cells and immunohistochemically by an intense staining for the connective tissue glycoprotein fibronectin (FN). Benign breast tissue displayed a universal, intense cytoplasmic but no extracellular reaction for TN, with the exception of rare foci of granulation tissue and around dilated cysts. Functional studies have shown that human embryonal lung fibroblasts increase their release of TN to the growth medium upon stimulation. The presence of TN extracellularly within fibroblast-rich foci of desmoplasia (and granulation tissue) suggests that a similar increased release of the protein takes place in vivo during active states. Desmoplasia has been found to have a protective effect on tumor cell propagation and metastasis in a murine model. The molecular interactions, which are responsible for this effect, are undoubtedly complex. However, TN may, by its specific binding to kringle 4 of plasminogen and its high affinity for sulphated polysaccharides, add to the understanding of how plasminogen activation is modulated at the local extracellular level.     

Cathepsin L increases invasion and migration of B16 melanoma

Background  

Most cancers express elevated protease levels which contribute to certain aspects of tumor behavior such as growth, metastatic spread, and angiogenesis. Elevation of the cathepsins of the cysteine protease family correlates with increased invasion of tumor cells. Cysteine proteases such as cathepsins B, H and L type participate in tumor cell invasion as extracellular proteases, yet are enzymes whose exact roles in metastasis are still being elucidated.     

Mechanical control of spheroid growth: distinct morphogenetic regimes

We develop a model of transport and growth in epithelio-mesenchymal interactions. Analysis of the growth of an avascular solid spheroid inside a passive mesenchyme or gel shows that sustained volumetric growth requires four generic mechanisms: (1) growth factor, (2) protease, (3) control of cellularity, and (4) swelling. The model reveals a bifurcation delineating two distinct morphogenetic regimes: (A) steady growth, (B) growth arrested by capsule formation in the mesenchyme. In both morphogenetic regimes, growth velocity is constant unless and until a complete capsule forms. Comprehensive exploration of the large parameter space reveals that the bifurcation is determined by just two ratios representing the relative strengths of growth and proteolytic activity. Growth velocity is determined only by the ratio governing growth, independent of proteolytic activity. There is a continuum of interior versus surface growth, with fastest growth at the surface. The model provides a theoretical basis for explaining observations of growth arrest despite proteolysis of surrounding tissue, and gives a quantitative framework for the design and interpretation of experiments involving spheroids, and tissues which are locally equivalent to spheroids.     

Interfiber tension transmission in series-fibered muscles of the cat hindlimb

Several muscles of the cat hindlimb, including biceps femoris and tenuissimus, are composed of short, in-series muscle fibers with tapered intrafascicular terminations. Tension generation and transmission within such muscles requires that active fibers should be mechanically coupled in series via myomyous junctions, specialized connective tissue attachments, or the endomysium. This report establishes that the tapered fibers of the cat biceps femoris and tenuissimus muscles have insignificant numbers of either myomyous or specialized connective tissue junctions. Tension appears to be transmitted in a distributed manner across the plasmalemma of the tapered (and probably the non-tapered) portions of the fibers to the connective tissue of the endomysium, which is therefore an essential series elastic element in these muscles. Subplasmalemmal dense plaques were identified and may play a role in transmembrane force transmission. In addition to the endomysium, passive muscle fibers may also serve to transmit tension between active fibers, and therefore should also be considered to be series elastic elements.