Co-implanting orthotopic tissue creates stroma microenvironment enhancing growth and angiogenesis of multiple tumors.

Tumor models are needed to study cancer. Noninvasive imaging of tumors under native conditions in vivo is critical but challenging. Intravital microscopy (IVM) of subcutaneous tumors provides dynamic, continuous, long-term imaging at high resolution. Although popular, subcutaneous tumor models are often criticized for being ectopic and lacking orthotopic tissue microenvironments critical for proper development. Similar IVM of orthotopic and especially spontaneous tumors is seldom possible. Here, we generate and characterize tumor models in mice for breast, lung, prostate and ovarian cancer by co-engrafting tumor spheroids with orthotopic tissue in dorsal skin window chambers for IVM. We use tumor cells and tissue, both genetically engineered to express distinct fluorescent proteins, in order to distinguish neoplastic cells from engrafted tissue. IVM of this new, two-colored model reveals classic tumor morphology with red tumor cell nests surrounded by green stromal elements. The co-implanted tissue forms the supportive stroma and vasculature of these tumors. Tumor growth and angiogenesis are more robust when tumor cells are co-implanted with orthotopic tissue versus other tissues, or in the skin alone. The orthotopic tissue promotes tumor cell mitosis over apoptosis. With time, tumor cells can adapt to new environments and ultimately even grow better in the non-orthotopic tissue over the original orthotopic tissue. These models offer a significant advance by recreating an orthotopic microenvironment in an ectopic location that is still easy to image by IVM. These "ectopic-orthotopic" models provide an exceptional way to study tumor and stroma cells in cancer, and directly show the critical importance of microenvironment in the development of multiple tumors.

Designed auto-assembly of nanostreptabodies for rapid tissue-specific targeting in vivo.

Molecular medicine can benefit greatly from antibodies that deliver therapeutic and imaging agents to select organs and diseased tissues. Yet the development of complex and defined composite nanostructures remains a challenge that requires both designed stoichiometric assembly and superior in vivo testing ability. Here, we generate nanostructures called nanostreptabodies by controlled sequential assembly of biotin-engineered antibody fragments on a streptavidin scaffold with a defined capacity for additional biotinylated payloads such as other antibodies to create bispecific antibodies as well as organic and non-organic moieties. When injected intravenously, these novel and stable nanostructures exhibit exquisite targeting with tissue-specific imaging and delivery, including rapid transendothelial transport that enhances tissue penetration. This "tinkertoy construction" strategy provides a very flexible and efficient way to link targeting vectors with reporter and/or effector agents, thereby providing virtually endless combinations potentially useful for multipurpose molecular and functional imaging in vivo as well as therapies.

Immunotargeting and cloning of two CD34 variants exhibiting restricted expression in adult rat endothelia in vivo.

Mapping protein expression of endothelial cells (EC) in vivo is fundamental to understanding cellular function and may yield new tissue-selective targets. We have developed a monoclonal antibody, MAb J120, to a protein expressed primarily in rat lung and heart endothelium. The antigen was identified as CD34, a marker of hematopoietic stem cells and global marker of endothelial cells in human and mouse tissues. PCR-based cloning identified two CD34 variant proteins, full length and truncated, both of which are expressed on luminal endothelial cell plasma membranes (P) isolated from lung. Truncated CD34 predominated in heart P, and neither variant was detected in P from kidney or liver. CD34 in lung was readily accessible to (125)I-J120 inoculated intravenously, and immunohistochemistry showed strong CD34 expression in lung EC. Few microvessels stained in heart and kidney, and no CD34 was detected in vessels of other organs or in lymphatics. We present herein the first complete sequence of a rat CD34 variant and show for the first time that the encoded truncated variant is endogenously expressed on EC in vivo. We also demonstrate that CD34 expression in rat EC, unlike mouse and human, is restricted in its distribution enabling quite specific lung targeting in vivo.

Neutral sphingomyelinase inhibitor scyphostatin prevents and ceramide mimics mechanotransduction in vascular endothelium.

Recently, we showed that neutral sphingomyelinase (N-SMase) is concentrated at the endothelial cell surface in caveolae and is activated to produce ceramide in an acute and transient manner by increase in flow rate and pressure in rat lung vasculature (Czarny M, Liu J, Oh P, and Schnitzer JE, J Biol Chem 278: 4424-4430, 2003). Here, we report further on our investigations of this new acute mechanotransduction pathway. We employed three experimental models to explore the role of N-SMase and ceramides in mechanosignaling: 1) a cell-free, in vitro model using isolated luminal plasma membranes of rat lung endothelium; 2) a fluid shear stress model using monolayers of intact bovine aorta endothelial cell in culture; and 3) an in situ model using controlled perfusion of the rat lung vasculature. Scyphostatin, which specifically inhibited N-SMase but not acid SMase activity, prevented mechanoactivation of N-SMase as well as downstream tyrosine and mitogen-activated protein kinases. Cell-permeable ceramide analogs (N-acetylsphingosine, C2-ceramide, and N-hexanoylsphingosine, C6-ceramide) but not the inactive dihydroderivatives D2-ceramide and D6-ceramide (N-acetylsphinganine and N-hexanoylsphinganine, respectively) mimic rapid mechano-induced tyrosine phosphorylation of cell surface proteins as well as mechanoactivation of Src-like kinases and the extracellular regulated kinase pathway. The responses common to ceramide and mechanical stress were inhibited by genistein, herbamycin A, and PP2, but not PP3, which suggests an obligate role of Src-like kinases in ceramide-mediated mechanotransduction. Ceramides also induced serine/threonine phosphorylation to activate the Akt/endothelial nitric oxide synthase pathway. Thus N-SMase at the plasma membrane in caveolae may be an upstream initiating mechanosensor, which acutely triggers mechanotransduction by generation of the lipid second messenger ceramide.

Transient mechanoactivation of neutral sphingomyelinase in caveolae to generate ceramide.

The vascular endothelium acutely autoregulates blood flow in vivo in part through unknown mechanosensing mechanisms. Here, we report the discovery of a new acute mechanotransduction pathway. Hemodynamic stressors from increased vascular flow and pressure in situ rapidly and transiently induce the activity of neutral sphingomyelinase but not that acid sphingomyelinase in a time- and flow rate-dependent manner, followed by the generation of ceramides. This acute mechanoactivation occurs directly at the luminal endothelial cell surface primarily in caveolae enriched in sphingomyelin and neutral sphingomyelinase, but not acid sphingomyelinase. Scyphostatin, which specifically blocks neutral but not acid sphingomyelinase, inhibits mechano-induced neutral sphingomyelinase activity as well as downstream activation of extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) by increased flow in situ. We postulate a novel physiological function for neutral sphingomyelinase as a new mechanosensor initiating the ERK cascade and possibly other mechanotransduction pathways.

[Repair of DNA damage using nucleotide excision repair (NER)--relationship with cancer risk]. / Naprawa uszkodzen DNA przez wycinanie nukleotydów (NER)--zwiazek z ryzykiem zachorowania na nowotwory.

DNA damage repair, responsible for maintaining the genome integrity, plays a central role in cancer biology. Individual DNA repair capacity is genetically determined. Inherited defect in nucleotide excision repair (NER) genes leads to three distinct and extremely rare disorders: xeroderma pigmentosum, associated with high risk of skin cancer, Cockayne syndrome, and trichothiodystrophy. The recently identified common polymorphism in several NER genes may also influence a risk of cancer in general population. The review presents current knowledge about a role of genetic variation of NER genes in cancer predisposition.