In vivo optical imaging of tumor stromal cells with hypoxia-inducible factor activity.

Tumors contain various stromal cells, such as immune cells, endothelial cells, and fibroblasts, which contribute to the development of a tumor-specific microenvironment characterized by hypoxia and inflammation, and are associated with malignant progression. In this study, we investigated the activity of intratumoral hypoxia-inducible factor (HIF), which functions as a master regulator of the cellular response to hypoxia and inflammation. We constructed the HIF activity-monitoring reporter gene hypoxia-response element-Venus-Akaluc (HVA) that expresses the green fluorescent protein Venus and modified firefly luciferase Akaluc in a HIF activity-dependent manner, and created transgenic mice harboring HVA transgene (HVA-Tg). In HVA-Tg, HIF-active cells can be visualized using AkaBLI, an ultra-sensitive in vivo bioluminescence imaging technology that produces an intense near-infrared light upon reaction of Akaluc with the D-luciferin analog AkaLumine-HCl. By orthotopic transplantation of E0771, a mouse triple negative breast cancer cell line without a reporter gene, into HVA-Tg, we succeeded in noninvasively monitoring bioluminescence signals from HIF-active stromal cells as early as 8 days after transplantation. The HIF-active stromal cells initially clustered locally and then spread throughout the tumors with growth. Immunohistochemistry and flow cytometry analyses revealed that CD11b+ F4/80+ macrophages were the predominant HIF-active stromal cells in E0771 tumors. These results indicate that HVA-Tg is a useful tool for spatiotemporal analysis of HIF-active tumor stromal cells, facilitating investigation of the roles of HIF-active tumor stromal cells in tumor growth and malignant progression.

Intraperitoneal transfer of microRNA-29b-containing small extracellular vesicles can suppress peritoneal metastases of gastric cancer.

Small extracellular vesicles (sEV) contain various microRNAs (miRNAs) and play crucial roles in the tumor metastatic process. Although miR-29b levels in peritoneal exosomes were markedly reduced in patients with peritoneal metastases (PM), their role has not been fully clarified. In this study, we asked whether the replacement of miR-29b can affect the development of PM in a murine model. UE6E7T-12, human bone marrow-derived mesenchymal stem cells (BMSCs), were transfected with miR-29b-integrating recombinant lentiviral vector and sEV were isolated from culture supernatants using ultracentrifugation. The sEV contained markedly increased amounts of miR-29b compared with negative controls. Treatment with transforming growth factor-ß1 decreased the expression of E-cadherin and calretinin with increased expression of vimentin and fibronectin on human omental tissue-derived mesothelial cells (HPMCs). However, the effects were totally abrogated by adding miR-29b-rich sEV. The sEV inhibited proliferation and migration of HPMCs by 15% (p < 0.005, n = 6) and 70% (p < 0.005, n = 6), respectively, and inhibited adhesion of NUGC-4 and MKN45 to HPMCs by 90% (p < 0.0001, n = 5) and 77% (p < 0.0001, n = 5), respectively. MicroRNA-29b-rich murine sEV were similarly obtained using mouse BMSCs and examined for in vivo effects with a syngeneic murine model using YTN16P, a highly metastatic clone of gastric cancer cell. Intraperitoneal (IP) transfer of the sEV every 3 days markedly reduced the number of PM from YTN16P in the mesentery (p < 0.05, n = 6) and the omentum (p < 0.05, n = 6). Bone marrow mesenchymal stem cell-derived sEV are a useful carrier for IP administration of miR-29b, which can suppress the development of PM of gastric cancer.

Design, synthesis, and evaluation of indeno[2,1-c]pyrazolones for use as inhibitors against hypoxia-inducible factor (HIF)-1 transcriptional activity.

HIF-1 is regarded as a promising target for the drugs used in cancer chemotherapy, and creating readily accessible templates for the development of synthetic drug candidates that could inhibit HIF-1 transcriptional activity is an important pursuit. In this study, indeno[2,1-c]pyrazolones were designed as readily available synthetic inhibitors of HIF-1 transcriptional activity. Nine compounds were synthesized in 4-5 steps from commercially available starting materials. In evaluations of the ability to inhibit the hypoxia-induced transcriptional activity of HIF-1, compound 3c showed a higher level compared with that of known inhibitor, YC-1. The compound 3c suppressed HIF-1α protein accumulation without affecting the levels of HIF-1α mRNA.

Novel lymphoid enhancer-binding factor 1-cytoglobin axis promotes extravasation of osteosarcoma cells into the lungs.

Lung metastasis is a major cause of mortality in patients with osteosarcoma (OS). A better understanding of the molecular mechanism of OS lung metastasis may facilitate development of new therapeutic strategies to prevent the metastasis. We have established high- and low-metastatic sublines (LM8-H and LM8-L, respectively) from Dunn OS cell line LM8 by using in vivo image-guided screening. Among the genes whose expression was significantly increased in LM8-H compared to LM8-L, the transcription factor lymphoid enhancer-binding factor 1 (LEF1) was identified as a factor that promotes LM8-H cell extravasation into the lungs. To identify downstream effectors of LEF1 that are involved in OS lung metastasis, 13 genes were selected based on LM8 microarray data and genomewide meta-analysis of a public database for OS patients. Among them, the cytoglobin (Cygb) gene was identified as a key effector in promoting OS extravasation into the lungs. CYGB overexpression increased the extravasation ability of LM8-L cells, whereas knocking out the Cygb gene in LM8-H cells reduced this ability. Our results showed a novel LEF1-CYGB axis in OS lung metastasis and may provide a new way of developing therapeutic strategies to prevent OS lung metastasis.

Hypoxia-inducible factor-targeting prodrug TOP3 combined with gemcitabine or TS-1 improves pancreatic cancer survival in an orthotopic model.

Pancreatic cancer is one of the most lethal digestive system cancers with a 5-year survival rate of 4-7%. Despite extensive efforts, recent chemotherapeutic regimens have provided only limited benefits to pancreatic cancer patients. Gemcitabine and TS-1, the current standard-of-care chemotherapeutic drugs for treatment of this severe cancer, have a low response rate. Hypoxia is one of the factors contributing to treatment resistance. Specifically, overexpression of hypoxia-inducible factor, a master transcriptional regulator of cell adaption to hypoxia, is strongly correlated with poor prognosis in many human cancers. TAT-ODD-procaspase-3 (TOP3) is a protein prodrug that is specifically processed and activated in hypoxia-inducible factor-active cells in cancers, leading to cell death. Here, we report combination therapies in which TOP3 was combined with gemcitabine or TS-1. As monotherapy, gemcitabine and TS-1 showed a limited effect on hypoxic and starved pancreatic cancer cells, whereas co-treatment with TOP3 successfully overcame this limitation in vitro. Furthermore, combination therapies of TOP3 with these drugs resulted in a significant improvement in survival of orthotopic pancreatic cancer models involving the human pancreatic cancer cell line SUIT-2. Overall, our study indicates that the combination of TOP3 with current chemotherapeutic drugs can significantly improve treatment outcome, offering a promising new therapeutic option for patients with pancreatic cancer.

Preparation of a cross-linked porous protein crystal containing Ru carbonyl complexes as a CO-releasing extracellular scaffold.

Protein crystals generally are stable solid protein assemblies. Certain protein crystals are suitable for use as nanovessels for immobilizing metal complexes. Here we report the preparation of ruthenium carbonyl-incorporated cross-linked hen egg white lysozyme crystals (Ru·CL-HEWL). Ru·CL-HEWL retains a Ru carbonyl moiety that can release CO, although a composite of Ru carbonyl-HEWL dissolved in buffer solution (Ru·HEWL) does not release CO. We found that treatment of cells with Ru·CL-HEWL significantly increased nuclear factor kappa B (NF-κB) activity as a cellular response to CO. These results demonstrate that Ru·CL-HEWL has potential for use as an artificial extracellular scaffold suitable for transport and release of a gas molecule.

Intracellular CO release from composite of ferritin and ruthenium carbonyl complexes.

Protein cages have been utilized as templates in the development of biomaterials. Here we report protein engineering of the ferritin (Fr) cage for encapsulating carbon monoxide releasing molecules (CORMs) and release of CO gas which serves as a cell signaling molecule. The protein cages enable us to increase the half-life for CO release, providing a release rate that is 18-fold slower than the rate of a typical CORM, Ru(CO)3Cl(glycinate) (CORM-3). Moreover, the uptake ratio of the composite is about 4-fold greater than that of CORM-3. We found that these effects enhance the activation of nuclear factor κB 10-fold higher than CORM-3. The protein cage of Fr thus provides the basis for new CORMs that can be used for in vitro cell research.

Bone resorption facilitates osteoblastic bone metastatic colonization by cooperation of insulin-like growth factor and hypoxia.

Bone metastasis is a multistep process that includes cancer cell dissemination, colonization, and metastatic growth. Furthermore, this process involves complex, reciprocal interactions between cancer cells and the bone microenvironment. Bone resorption is known to be involved in both osteolytic and osteoblastic bone metastasis. However, the precise roles of the bone resorption in the multistep process of osteoblastic bone metastasis remain unidentified. In this study, we show that bone resorption plays important roles in cancer cell colonization during the initial stage of osteoblastic bone metastasis. We applied bioluminescence/X-ray computed tomography multimodal imaging that allows us to spatiotemporally analyze metastasized cancer cells and bone status in osteoblastic bone metastasis models. We found that treatment with receptor activator of factor-κB ligand (RANKL) increased osteoblastic bone metastasis when given at the same time as intracardiac injection of cancer cells, but failed to increase metastasis when given 4 days after cancer cell injection, suggesting that RANKL-induced bone resorption facilitates growth of cancer cells colonized in the bone. We show that insulin-like growth factor-1 released from the bone during bone resorption and hypoxia-inducible factor activity in cancer cells cooperatively promoted survival and proliferation of cancer cells in bone marrow. These results suggest a mechanism that bone resorption and hypoxic stress in the bone microenvironment cooperatively play an important role in establishing osteoblastic metastasis.

MT1-MMP plays a critical role in hematopoiesis by regulating HIF-mediated chemokine/cytokine gene transcription within niche cells.

HSC fate decisions are regulated by cell-intrinsic and cell-extrinsic cues. The latter cues are derived from the BM niche. Membrane-type 1 matrix metalloproteinase (MT1-MMP), which is best known for its proteolytic role in pericellular matrix remodeling, is highly expressed in HSCs and stromal/niche cells. We found that, in MT1-MMP(-/-) mice, in addition to a stem cell defect, the transcription and release of kit ligand (KitL), stromal cell-derived factor-1 (SDF-1/CXCL12), erythropoietin (Epo), and IL-7 was impaired, resulting in a trilineage hematopoietic differentiation block, while addition of exogenous KitL and SDF-1 restored hematopoiesis. Further mechanistic studies revealed that MT1-MMP activates the hypoxia-inducible factor-1 (HIF-1) pathway via factor inhibiting HIF-1 (FIH-1) within niche cells, thereby inducing the transcription of HIF-responsive genes, which induce terminal hematopoietic differentiation. Thus, MT1-MMP in niche cells regulates postnatal hematopoiesis, by modulating hematopoietic HIF-dependent niche factors that are critical for terminal differentiation and migration.

A hypoxia-inducible factor (HIF)-3α splicing variant, HIF-3α4 impairs angiogenesis in hypervascular malignant meningiomas with epigenetically silenced HIF-3α4.

Hypoxia inducible factor is a dominant regulator of adaptive cellular responses to hypoxia and controls the expression of a large number of genes regulating angiogenesis as well as metabolism, cell survival, apoptosis, and other cellular functions in an oxygen level-dependent manner. When a neoplasm is able to induce angiogenesis, tumor progression occurs more rapidly because of the nutrients provided by the neovasculature. Meningioma is one of the most hypervascular brain tumors, making anti-angiogenic therapy an attractive novel therapy for these tumors. HIF-3α has been conventionally regarded as a dominant-negative regulator of HIF-1α, and although alternative HIF-3α splicing variants are extensively reported, their specific functions have not yet been determined. In this study, we found that the transcription of HIF-3α4 was silenced by the promoter DNA methylation in meningiomas, and inducible HIF-3α4 impaired angiogenesis, proliferation, and metabolism/oxidation in hypervascular meningiomas. Thus, HIF-3α4 could be a potential molecular target in meningiomas.

Secretory GFP reconstitution labeling of neighboring cells interrogates cell-cell interactions in metastatic niches.

Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

Depth estimation of tumor invasion in early gastric cancer using scattering of circularly polarized light: Monte Carlo Simulation study.

Quantitative depth estimation of tumor invasion in early gastric cancer by scattering of circularly polarized light is computationally investigated using the Monte Carlo method. Using the optical parameters of the human stomach wall and its carcinoma, the intensity and circular polarization of light scattered from pseudo-healthy and cancerous tissues were calculated over a wide spectral range. Large differences in the circular polarization with opposite signs, together with the large intensity, are obtained at wavelengths 600 nm and 950 nm. At these two wavelengths, the sampling depth of the biological tissues can be modulated by tuning the detection angle. In bi-layered pseudo-tissues with a cancerous layer on a healthy layer and vice versa, the degree of circular polarization of scattered light shows systematic changes depending on the thickness and depth of the cancerous layer, which indicates the feasibility of in vivo quantitative estimation of cancer progression in early gastric cancer.

The role of MerTK in promoting cell migration is enhanced by the oncogenic Ras/IL-33 signaling axis.

Ras genes are frequently mutated in many cancer types; however, there are currently no conclusively effective anticancer drugs against Ras-induced cancer. Therefore, the downstream effectors of Ras signaling need to be identified for the development of promising novel therapeutic approaches. We previously reported that oncogenic Ras induced the expression of NF-HEV/IL-33, a member of the interleukin-1 family, and showed that intracellular IL-33 was required for oncogenic Ras-induced cellular transformation. In the present study, we demonstrated that the c-Mer proto-oncogene tyrosine kinase (MerTK), a receptor tyrosine kinase, played essential roles in oncogenic Ras/IL-33 signaling. The expression of MerTK was enhanced in transformed NIH-3T3 cells by the expression of oncogenic Ras, H-Ras (G12V), in an IL-33-dependent manner. In human colorectal cancer tissues, MerTK expression also correlated with IL-33 expression. The knockdown of IL-33 or MerTK effectively attenuated the migration of NIH-3T3 cells transformed by H-Ras (G12V) and A549, LoVo, and HCT116 cells harboring an oncogenic K-Ras mutation. Furthermore, the suppression of Ras-induced cell migration by the knockdown of IL-33 was rescued by the enforced expression of MerTK. The present results also revealed that MerTK was effectively phosphorylated in NIH-3T3 cells transformed by Ras (G12V). Ras signaling was essential for the tyrosine phosphorylation of MerTK, and the kinase activity of MerTK was indispensable for accelerating cell migration. Collectively, the present results reveal a novel role for MerTK in cancer malignancy, which may be utilized to develop novel therapeutic strategies that target Ras-transformed cells.

AGE/RAGE axis regulates reversible transition to quiescent states of ALK-rearranged NSCLC and pancreatic cancer cells in monolayer cultures.

Cancer recurrence due to tumor cell quiescence after therapy and long-term remission is associated with cancer-related death. Previous studies have used cell models that are unable to return to a proliferative state; thus, the transition between quiescent and proliferative states is not well understood. Here, we report monolayer cancer cell models wherein the human non-small cell lung carcinoma cell line H2228 and pancreatic cancer cell line AsPC-1 can be reversibly induced to a quiescent state under hypoxic and serum-starved (HSS) conditions. Transcriptome and metabolome dual-omics profiles of these cells were compared with those of the human lung adenocarcinoma cell line A549, which was unable to enter a quiescent state under HSS conditions. The quiescence-inducible cells had substantially lower intracellular pyruvate and ATP levels in the quiescent state than in the proliferative state, and their response to sudden demand for energy was dramatically reduced. Furthermore, in quiescence-inducible cells, the transition between quiescent and proliferative states of these cells was regulated by the balance between the proliferation-promoting Ras and Rap1 signaling and the suppressive AGE/RAGE signaling. These cell models elucidate the transition between quiescent and proliferative states, allowing the development of drug-screening systems for quiescent tumor cells.

Pathophysiological response to hypoxia - from the molecular mechanisms of malady to drug discovery:hypoxia-inducible factor-1 (HIF-1)-active cells as a target for cancer therapy.

The microenvironment of solid tumors is characterized by low pO(2) that is well below physiological levels. Intratumoral hypoxia is a major factor contributing to cancer progression and is exacerbated as a result of oxygen consumption by rapidly proliferating tumor cells near blood vessels, poor lymphatic drainage resulting in high interstitial pressure, and irregular blood supply through immature tumor vasculature. Hypoxia-inducible factor-1 (HIF-1) is the main transcription factor that regulates cellular responses to hypoxia. Cellular changes induced by HIF-1 are extremely important targets for cancer therapy. Therefore, targeting strategies to counteract HIF-1-active cells are essential for cancer therapy. In this study, we introduce a novel strategy for targeting HIF-1-active cells.

A Murine Bone Metastasis Model Using Caudal Artery Injection and Bioluminescence Imaging.

The current standard murine model of bone metastasis by using intracardiac injection (IC) has some limitations despite the great utility of this model. This fact emphasizes the need for a new murine model to accelerate basic research of bone metastasis. The present protocol provides instructions on caudal artery (CA) injection that is an easy-to-use method to reliably construct a murine bone metastasis model with a variety type of cancer cell lines. Bioluminescence imaging visualized that cancer cells injected via the caudal artery in the tail were efficiently delivered to a hind limb bone, where it is a common site affected with bone metastasis in mice. CA injection rarely causes stress-induced acute death in mice and enables us to inject a large number of cancer cells, thereby greatly increasing the frequency of bone metastasis in hind limb bones. Importantly, CA injection is technically as easy as tail vein injection and causes no lethal stress, indicating that it is a model that also contributes to animal welfare. CA injection model, therefore, could represent a powerful tool for many researchers to study molecular mechanisms of bone metastasis in mice.

Angular optimization for cancer identification with circularly polarized light.

Depolarization of circularly polarized light scattered from biological tissues depends on structural changes in cell nuclei, which can provide valuable information for differentiating cancer tissues concealed in healthy tissues. In this study, we experimentally verified the possibility of cancer identification using scattering of circularly polarized light. We investigated the polarization of light scattered from a sliced biological tissue with various optical configurations. A significant difference between circular polarizations of light scattered from cancerous and healthy tissues is observed, which is sufficient to distinguish a cancerous region. The line-scanning experiments along a region incorporating healthy and cancerous parts indicate step-like behaviors in the degree of circular polarization corresponding to the state of tissues, whether cancerous or normal. An oblique and perpendicular incidence induces different resolutions for identifying cancerous tissues, which indicates that the optical arrangement can be selected according to the priority of resolution.

Agonist dependency of the second phase access of ß-arrestin 2 to the heteromeric µ-V1b receptor.

During the development of analgesic tolerance to morphine, the V1b vasopressin receptor has been proposed to bind to ß-arrestin 2 and the µ-opioid receptor to enable their interaction. However, direct evidence of such a high-order complex is lacking. Using bioluminescent resonance energy transfer between a split Nanoluciferase and the Venus fluorescent protein, the NanoBit-NanoBRET system, we found that ß-arrestin 2 closely located near the heteromer µ-V1b receptor in the absence of an agonist and moved closer to the receptor carboxyl-termini upon agonist stimulation. An additive effect of the two agonists for opioid and vasopressin receptors was detected on the NanoBRET between the µ-V1b heteromer and ß-arrestin 2. To increase the agonist response of NanoBRET, the ratio of the donor luminophore to the acceptor fluorophore was decreased to the detection limit of luminescence. In the first phase of access, ß-arrestin 2 was likely to bind to the unstimulated V1b receptor in both its phosphorylated and unphosphorylated forms. In contrast, the second-phase access of ß-arrestin 2 was agonist dependent, indicating a possible pharmacological intervention strategy. Therefore, our efficient method should be useful for evaluating chemicals that directly target the vasopressin binding site in the µ-V1b heteromer to reduce the second-phase access of ß-arrestin 2 and thereby to alleviate tolerance to morphine analgesia.

Antibody-guided design and identification of CD25-binding small antibody mimetics using mammalian cell surface display.

Small antibody mimetics that contain high-affinity target-binding peptides can be lower cost alternatives to monoclonal antibodies (mAbs). We have recently developed a method to create small antibody mimetics called FLuctuation-regulated Affinity Proteins (FLAPs), which consist of a small protein scaffold with a structurally immobilized target-binding peptide. In this study, to further develop this method, we established a novel screening system for FLAPs called monoclonal antibody-guided peptide identification and engineering (MAGPIE), in which a mAb guides selection in two manners. First, antibody-guided design allows construction of a peptide library that is relatively small in size, but sufficient to identify high-affinity binders in a single selection round. Second, in antibody-guided screening, the fluorescently labeled mAb is used to select mammalian cells that display FLAP candidates with high affinity for the target using fluorescence-activated cell sorting. We demonstrate the reliability and efficacy of MAGPIE using daclizumab, a mAb against human interleukin-2 receptor alpha chain (CD25). Three FLAPs identified by MAGPIE bound CD25 with dissociation constants of approximately 30 nM as measured by biolayer interferometry without undergoing affinity maturation. MAGPIE can be broadly adapted to any mAb to develop small antibody mimetics.

Calcium phosphate microcrystals in the renal tubular fluid accelerate chronic kidney disease progression.

The Western pattern diet is rich not only in fat and calories but also in phosphate. The negative effects of excessive fat and calorie intake on health are widely known, but the potential harms of excessive phosphate intake are poorly recognized. Here, we show the mechanism by which dietary phosphate damages the kidney. When phosphate intake was excessive relative to the number of functioning nephrons, circulating levels of FGF23, a hormone that increases the excretion of phosphate per nephron, were increased to maintain phosphate homeostasis. FGF23 suppressed phosphate reabsorption in renal tubules and thus raised the phosphate concentration in the tubule fluid. Once it exceeded a threshold, microscopic particles containing calcium phosphate crystals appeared in the tubule lumen, which damaged tubule cells through binding to the TLR4 expressed on them. Persistent tubule damage induced interstitial fibrosis, reduced the number of nephrons, and further boosted FGF23 to trigger a deterioration spiral leading to progressive nephron loss. In humans, the progression of chronic kidney disease (CKD) ensued when serum FGF23 levels exceeded 53 pg/mL. The present study identified calcium phosphate particles in the renal tubular fluid as an effective therapeutic target to decelerate nephron loss during the course of aging and CKD progression.