Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.
Tumor hypoxia has been considered to be a potential therapeutic problem because it renders solid tumors more resistant to sparsely ionizing radiation (IR) and chemotherapeutic drugs. Moreover, recent laboratory and clinical data have shown that tumor hypoxia is also associated with a more malignant phenotype and poor survival in patients suffering from various solid tumors. Therefore, selective targeting of hypoxic tumor cells has been explored, and since severe hypoxia (pO 2 < < < <0.33%, 2.5 mmHg) does not occur in normal tissue, tumor hypoxia could be exploited for therapeutic advantage. However, the following three characteristics of hypoxic tumor regions present obstacles in targeting hypoxic cells. First, it is difficult to deliver a sufficient amount of drug to a region that is remote from blood vessels. Second, one must specifically target hypoxic tumor cells while sparing normal well-oxygenated tissue from damage. Finally, the severely hypoxic tumor cells to be attacked have often stopped dividing. Therefore, high delivery efficiency, high specificity and selective cytotoxicity are all necessary to target and combat hypoxic tumor cells. The current review describes progress on the biological aspects of tumor hypoxia and provides a compilation of the recent molecular approaches used to target hypoxic tumors. These approaches include our work with a unique hypoxia-targeting protein drug, TOP3, with which we have sought to address the above three difficulties. (Cancer Sci 2003; 94: 1021-1028) n solid tumors, oxygen delivery to the respiring neoplastic and stromal cells is frequently reduced or even abolished by deteriorating diffusion geometry, severe structural abnormalities of tumor microvessels, and damaged microcirculation.1) It has been known for some time that tumor cells can survive and even grow in such a deteriorated microenvironment. Moreover, as described in detail below, tumor hypoxia has been shown to be a cause of malignant transformation and a source of resistance to current cancer therapies. For these reasons, approaches to targeting hypoxic tumor cells have been sought over a period of years. Recent progress in understanding the molecular mechanisms of hypoxic response, and especially the discovery of a key molecule, Hypoxia Inducible Factor (HIF), as well as improvements in diagnostic techniques through which the tumor microenvironment can be assessed, has opened the door to a solution of this longstanding issue.
A tumor-specific microenvironment is characterized by hypoxia, in which oxygen tension is considerably lower than in normal tissues.
In preclinical cancer research, bioluminescence imaging with firefly luciferase and D-luciferin has become a standard to monitor biological processes both in vitro and in vivo. However, the emission maximum (λmax) of bioluminescence produced by D-luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λmax=677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with D-luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models.
Hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. The transcription factor hypoxia-inducible factor-1 (HIF-1) is a major regulator of adaptation to hypoxia and is implicated in the malignant progression of cancers. Here, we studied whether hypoxia and HIF-1 expression contribute to the development of bone metastases using a well-characterized animal model of bone metastasis in MDA-MB-231 human breast cancer cells. To study the role of hypoxia in bone metastases, we tested the effects of the fusion protein (TOP3), the oxygen-dependent degradation domain of HIF-1A fused with HIV-TAT, and procaspase-3. TOP3 selectively induced apoptosis in hypoxic tumor cells in vitro and significantly reduced bone metastases in vivo. We next examined the role of HIF-1 in bone metastases by establishing MDA-MB-231 cells overexpressing constitutively active or dominant-negative HIF-1A (MDA/CA-HIF or MDA/DN-HIF, respectively). Bone metastases of MDA/CA-HIF were significantly increased with elevated number of CD31-positive blood vessels. In contrast, bone metastases were significantly reduced in MDA/DN-HIF. Because the progression of osteolytic bone metastases is due in part to the imbalance between bone formation and bone resorption, we examined the effects of hypoxia and HIF-1 on the differentiation of osteoblasts and osteoclasts. Hypoxia and CA-HIF overexpression markedly inhibited osteoblastic differentiation, whereas hypoxia increased osteoclast-like cell formation. In conclusion, these results suggest that tumor-associated hypoxia and HIF-1 expression promote the progression of bone metastases in breast cancer. Our results also suggest that hypoxia and HIF-1 lead to the development of osteolytic bone metastases by suppressing osteoblast differentiation and promoting osteoclastogenesis. [Cancer Res 2007;67(9):4157-63]
Transcriptional activity of HIF-1 (hypoxia-inducible factor-1) has been reported to be up-regulated in solid tumors after ionizing radiation; however, the molecular mechanism underlying the response remains to be elucidated. In the present study, we performed a series of molecular imaging experiments using a HIF-1-dependent reporter gene, 5HREp-ODD-luc, and found an essential role of the Akt/mTOR pathway.
Recent reports have suggested critical roles of myeloid cells in tumor invasion and metastasis, although these findings have not led to therapeutics. Using a mouse model for liver dissemination, we show that mouse and human colon cancer cells secrete CCchemokine ligands CCL9 and CCL15, respectively, and recruit CD34 + Gr-1 − immature myeloid cells (iMCs). They express CCL9/15 receptor CCR1 and produce matrix metalloproteinases MMP2 and MMP9. Lack of the Ccr1, Mmp2, or Mmp9 gene in the host dramatically suppresses outgrowths of disseminated tumors in the liver. Importantly, CCR1 antagonist BL5923 blocks the iMC accumulation and metastatic colonization and significantly prolongs the survival of tumor-bearing mice. These results suggest that CCR1 antagonists can provide antimetastatic therapies for patients with disseminated colon cancer in the liver.C olon cancer is one of the leading causes of cancer-related deaths (1). Although most primary tumors can be resected surgically, colorectal cancer frequently spreads to the liver, which is responsible for the high mortality of the disease (2). For successful metastasis, cancer cells need to invade surrounding tissues, penetrate microvessels, survive in circulation, disseminate to distant organs, form micrometastases, and expand into macrometastases. To progress through these steps, tumor cells often acquire the capability of survival and invasion by activating metastatic signaling pathways or inactivating metastasis suppressor genes (2, 3). In addition to these cell autonomous changes, tumor stromal cells, especially bone marrow-derived myeloid cells, actively participate in early steps of the metastatic cascade in some mouse models (4). For example, tumor-associated macrophages (TAMs) promote migration and intravasation of mammary tumor cells (5, 6). Bone marrow-derived cells that express myeloid cell marker CD11b and granulocyte marker Gr-1 (CD11b + Gr-1 + ) also promote metastasis of breast cancer cells, likely through promotion of intravasation and suppression of immune responses (7). Furthermore, CD11b + myeloid cells that express vascular endothelial growth factor receptor 1 (VEGFR1) accumulate at the metastatic sites before the arrival of lung cancer and melanoma cells and foster the dissemination of the cancer cells (8). These reports suggest that bone marrow-derived myeloid cells can help cancer epithelium in early steps of metastasis. It remains to be determined whether therapeutics targeting such myeloid cells can prevent cancer metastasis (9).As a model for invasive colon cancer, we previously constructed cis-Apc +/Δ716 Smad4 +/− (Apc/Smad4) mice that develop intestinal adenocarcinomas with marked invasions by loss of Apc and Smad4 tumor suppressor genes in the intestinal epithelium (10, 11). In the Apc/Smad4 tumors, we reported that the invading cancer epithelium is associated with immature myeloid cells (iMCs) that express myeloid progenitor cell marker CD34 and CD11b (12). Because these iMCs do not express Gr-1 or VEGFR1, they belong to a different subc...
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