Ruth J. Muschel

Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.

Metastasis is a frequent complication of cancer, yet the process through which circulating tumor cells form distant colonies is poorly understood. We have been able to observe the steps in early hematogenous metastasis by epifluorescence microscopy of tumor cells expressing green fluorescent protein in subpleural microvessels in intact, perfused mouse and rat lungs. Metastatic tumor cells attached to the endothelia of pulmonary pre-capillary arterioles and capillaries. Extravasation of tumor cells was rare, and it seemed that the transmigrated cells were cleared quickly by the lung, leaving only the endothelium-attached cells as the seeds of secondary tumors. Early colonies were entirely within the blood vessels. Although most models of metastasis include an extravasation step early in the process, here we show that in the lung, metastasis is initiated by attachment of tumor cells to the vascular endothelium and that hematogenous metastasis originates from the proliferation of attached intravascular tumor cells rather than from extravasated ones. Intravascular metastasis formation would make early colonies especially vulnerable to intravascular drugs, and this possibility has potential for the prevention of tumor cell attachment to the endothelium.

A Distinct Macrophage Population Mediates Metastatic Breast Cancer Cell Extravasation, Establishment and Growth

Background The stromal microenvironment and particularly the macrophage component of primary tumors influence their malignant potential. However, at the metastatic site the role of these cells and their mechanism of actions for establishment and growth of metastases remain largely unknown. Methodology/Principal Findings Using animal models of breast cancer metastasis, we show that a population of host macrophages displaying a distinct phenotype is recruited to extravasating pulmonary metastatic cells regardless of species of origin. Ablation of this macrophage population through three independent means (genetic and chemical) showed that these macrophages are required for efficient metastatic seeding and growth. Importantly, even after metastatic growth is established, ablation of this macrophage population inhibited subsequent growth. Furthermore, imaging of intact lungs revealed that macrophages are required for efficient tumor cell extravasation. Conclusion/Significance These data indicate a direct enhancement of metastatic growth by macrophages through their effects on tumor cell extravasation, survival and subsequent growth and identifies these cells as a new therapeutic target for treatment of metastatic disease.

Cancer stem cells from colorectal cancer-derived cell lines

Cancer stem cells (CSCs) are the subpopulation of cells within a tumor that can self-renew, differentiate into multiple lineages, and drive tumor growth. Here we describe a two-pronged approach for the identification and characterization of CSCs from colorectal cancer cell lines, using a Matrigel-based differentiation assay, and cell surface markers CD44 and CD24. About 20 to 30% of cells from the SW1222 cell line form megacolonies in Matrigel that have complex 3D structures resembling colonic crypts. The megacolonies’ capacity to self-renew in vitro is direct evidence that they contain the CSCs. Furthermore, just 200 cells from SW1222 megacolonies initiate tumors in NOD/SCID mice. We also showed that CD44+CD24+ cells enriched for colorectal CSCs in the HT29 and SW1222 cell lines, which can self-renew and reform all four CD44/CD24 subpopulations, are the most clonogenic in vitro and can initiate tumors in vivo. A single SW1222 CD44+CD24+ CSC, when grown in Matrigel, can form large megacolonies that differentiate into enterocyte, enteroendocrine, and goblet cell lineages. The HCT116 line does not differentiate or express CDX1, nor does it contain subpopulations of cells with greater tumor-forming capacity, suggesting that HCT116 contains mainly CSCs. However, forced expression of CDX1 in HCT116 leads to reduced clonogenicity and production of differentiating crypt-containing colonies, which can explain the selection for reduced CDX1 expression in many colorectal cancers. In summary, colorectal cancer cell lines contain subpopulations of CSCs, characterized by their cell surface markers and colony morphology, which can self-renew and differentiate into multiple lineages.

The molecular basis for cell cycle delays following ionizing radiation: a review

Exposure of a wide variety of cells to ionizing (X- or gamma-) irradiation results in a division delay which may have several components including a G1 block, a G2 arrest or an S phase delay. The G1 arrest is absent in many cell lines, and the S phase delay is typically seen following relatively high doses (> 5 Gy). In contrast, the G2 arrest is seen in virtually all eukaryotic cells and occurs following high and low doses, even under 1 Gy. The mechanism underlying the G2 arrest may involve suppression of cyclin B1 mRNA and/or protein in some cell lines and tyrosine phosphorylation of p34cdc2 in others. Similar mechanisms are likely to be operative in the G2 arrest induced by various chemotherapeutic agents including nitrogen mustard and etoposide. The upstream signal transduction pathways involved in the G2 arrest following ionizing radiation remain obscure in mammalian cells; however, in the budding yeast the rad9 gene and in the fission yeast the chk1/rad27 gene are involved. There is evidence indicating that shortening of the G2 arrest results in decreased survival which has led to the hypothesis that during this block, cells repair damaged DNA following exposure to genotoxic agents. In cell lines examined to date, wildtype p53 is required for the G1 arrest following ionizing radiation. The gadd45 gene may also have a role in this arrest. Elimination of the G1 arrest leads to no change in survival following radiation in some cell lines and increased radioresistance in others. It has been suggested that this induction of radioresistance in certain cell lines is due to loss of the ability to undergo apoptosis. Relatively little is known about the mechanism underlying the S phase delay. This delay is due to a depression in the rate of DNA synthesis and has both a slow and a fast component. In some cells the S phase delay can be abolished by staurosporine, suggesting involvement of a protein kinase. Understanding the molecular mechanisms behind these delays may lead to improvement in the efficacy of radiotherapy and/or chemotherapy if they can be exploited to decrease repair or increase apoptosis following exposure to those agents.

Coagulation Facilitates Tumor Cell Spreading in the Pulmonary Vasculature during Early Metastatic Colony Formation

Coagulation has long been known to facilitate metastasis. To pinpoint the steps where coagulation might play a role in the metastasis, we used three-dimensional visualization of direct infusion of fluorescence labeled antibody to observe the interaction of tumor cells with platelets and fibrinogen in isolated lung preparations. Tumor cells arrested in the pulmonary vasculature were associated with a clot composed of both platelets and fibrin(ogen). Initially, the cells attached to the pulmonary vessels were rounded. Over the next 2 to 6 hours, they spread on the vessel surface. The associated clot was lysed coincident with tumor cell spreading. To assess the importance of clot formation, we inhibited coagulation with hirudin, a potent inhibitor of thrombin. The number of tumor cells initially arrested in the lung of hirudin-treated mice was essentially the same as in control mice. However, tumor cell spreading and subsequent retention of the tumor cells in the lung was markedly inhibited in the anticoagulated mice. These associations of the tumor cells with platelets were independent of tumor cell expression of P-selectin ligands. This work identifies tumor cell spreading onto the vascular surface as an important component of the metastatic cascade and implicates coagulation in this process.

Proteases, Extracellular Matrix, and Cancer: A Workshop of the Path B Study Section

The role of the extracellular matrix (ECM) in the tumor microenvironment is not limited to being a barrier against tumor invasion. The ECM is a reservoir of cell binding proteins and growth factors that affect tumor cell behavior. It is also substantially modified by proteases produced by tumor cells or stroma cells. As a result of the activity of these proteases, cell-cell and cell-ECM interactions are altered, new biologically active ECM molecules are generated, and the bioavailability and activity of many growth factors, growth factor receptors, and cytokines are modified. ECM-degrading proteases also play a critical role in angiogenesis, where they can act as positive as well as negative regulators of endothelial cell proliferation and vascular morphogenesis. This review article summarizes some of the most relevant findings made over the recent years that were discussed at a workshop organized by the Path B Study Section of the National Institutes of Health in October 2002.

The Vascular Basement Membrane as “Soil” in Brain Metastasis

Brain-specific homing and direct interactions with the neural substance are prominent hypotheses for brain metastasis formation and a modern manifestation of Pagets “seed and soil” concept. However, there is little direct evidence for this “neurotropic” growth in vivo. In contrast, many experimental studies have anecdotally noted the propensity of metastatic cells to grow along the exterior of pre-existing vessels of the CNS, a process termed vascular cooption. These observations suggest the “soil” for malignant cells in the CNS may well be vascular, rather than neuronal. We used in vivo experimental models of brain metastasis and analysis of human clinical specimens to test this hypothesis. Indeed, over 95% of early micrometastases examined demonstrated vascular cooption with little evidence for isolated neurotropic growth. This vessel interaction was adhesive in nature implicating the vascular basement membrane (VBM) as the active substrate for tumor cell growth in the brain. Accordingly, VBM promoted adhesion and invasion of malignant cells and was sufficient for tumor growth prior to any evidence of angiogenesis. Blockade or loss of the β1 integrin subunit in tumor cells prevented adhesion to VBM and attenuated metastasis establishment and growth in vivo. Our data establishes a new understanding of CNS metastasis formation and identifies the neurovasculature as the critical partner for such growth. Further, we have elucidated the mechanism of vascular cooption for the first time. These findings may help inform the design of effective molecular therapies for patients with fatal CNS malignancies.

BRCA1 Effects on the Cell Cycle and the DNA Damage Response Are Linked to Altered Gene Expression

The breast and ovarian cancer susceptibility gene product BRCA1 has been reported to be expressed in a cell cycle-dependent manner; possess transcriptional activity; associate with several proteins, including the p53 tumor suppressor; and play an integral role in certain types of DNA repair. We show here that ectopic expression of BRCA1 using an adenovirus vector (Ad-BRCA1) leads to dephosphorylation of the retinoblastoma protein accompanied by a decrease in cyclin-dependent kinase activity. Flow cytometric analysis on Ad-BRCA1-infected cells revealed a G1 or G2 phase accumulation. High density cDNA array screening of colon, lung, and breast cancer cells identified several genes affected by BRCA1 expression in a p53-independent manner, including DNA damage response genes and genes involved in cell cycle control. Notable changes included induction of the GADD45 and GADD153 genes and a reduction in cyclin B1 expression. Therefore, BRCA1 has the potential to modulate the expression of genes and function of proteins involved in cell cycle control and DNA damage response pathways.

Tumor cell α3β1 integrin and vascular laminin-5 mediate pulmonary arrest and metastasis

Arrest of circulating tumor cells in distant organs is required for hematogenous metastasis, but the tumor cell surface molecules responsible have not been identified. Here, we show that the tumor cell α3β1 integrin makes an important contribution to arrest in the lung and to early colony formation. These analyses indicated that pulmonary arrest does not occur merely due to size restriction, and raised the question of how the tumor cell α3β1 integrin contacts its best-defined ligand, laminin (LN)-5, a basement membrane (BM) component. Further analyses revealed that LN-5 is available to the tumor cell in preexisting patches of exposed BM in the pulmonary vasculature. The early arrest of tumor cells in the pulmonary vasculature through interaction of α3β1 integrin with LN-5 in exposed BM provides both a molecular and a structural basis for cell arrest during pulmonary metastasis.

Radiation sensitization of human cancer cells in vivo by inhibiting the activity of PI3K using LY294002

Multiple genetic alterations such as in Ras or EGFR can result in sustained signaling through PI3K. Our previous experiments have shown that resistance to radiation results from PI3K activity in cells in culture. Here we examined whether inhibition of PI3K in vivo would sensitize tumors to radiation. The human bladder cancer cell line T24 has amplified and mutated H-Ras resulting in sustained PI3K activity and phosphorylation of the downstream target of PI3K, Akt. Nude mice bearing T24 tumor cell xenografts were randomly assigned to one of four groups: control, radiation alone, the PI3K inhibitor LY294002 alone, or combined LY294002 and radiation. The LY294002 was delivered intraperitoneally to the mice. Downregulation of Akt was documented by Western blot analysis of tumor lysates. In vivo sensitization was measured using clonogenic assays or regrowth assays.A dose of 100 mg/kg of LY294002, but not 50 mg/kg, consistently eliminated the phosphorylation of Akt. This inhibition was transient, and Akt activity returned after 30 min. This dose resulted in severe respiratory depression and lethargy resolving without lethality. It is not possible to tell whether these side effects of LY294002 were mechanism-based or idiosyncratic. The PI3K inhibitor LY294002 by itself had minimal antitumor effect. The combination of LY294002 and radiation resulted in significant and synergistic reduction in clonogenicity and growth delay. Inhibition of PI3K by LY294002 can synergistically enhance radiation efficacy. This acts as a proof of principle that inhibition of the Ras to PI3K pathway could be useful clinically.