Targeting Hypoxia Research Articles

Hypoxia-Targeting Multifunctional Nanoparticles for Sensitized Chemotherapy and Phototherapy in Head and Neck Squamous Cell Carcinoma.

PurposeChemotherapy in head and neck squamous cell carcinoma (HNSCC) has many systemic side effects, as well as hypoxia-induced chemoresistance. To reduce side effects and enhance chemosensitivity are urgently needed.MethodsWe synthesized a drug delivery system (named CECMa NPs) based on cisplatin (CDDP) and metformin (chemotherapeutic sensitizer), of which chlorin e6 (Ce6) and polyethylene glycol diamine (PEG) were synthesized as the shell, an anti-LDLR antibody (which can target to hypoxic tumor cells) was modified on the surface to achieve tumor targeting.ResultsThe NPs possessed a great synergistic effect of chemotherapy and phototherapy. After laser stimulation, both CDDP and metformin can be released in situ to achieve anti-tumor effects. Meanwhile, PDT and PTT triggered by a laser have anticancer effects. Furthermore, compared with free cisplatin, CECMa exhibits less systemic toxicity with laser irradiation in the xenograft mouse tumor model.ConclusionCECMa effectively destroyed the tumors via hypoxia targeting multimodal therapy both in vitro and in vivo, thereby providing a novel strategy for targeting head and neck squamous cell carcinoma.

Rational design of ERα targeting hypoxia turn-on fluorescent probes with antiproliferative activity for breast cancer.

The overexpression of estrogen receptor (ER) α is not only closely related to the development of ER+ breast cancer, but is also an important biomarker for clinical diagnosis and treatment. Herein, we report several ERα targeting hypoxia turn-on fluorescent probes with antitumor activity for breast cancer cells. Among them, probes 3 and 5 displayed good ERα targeting ability and favorable hypoxia turn-on response in MCF-7 cells. Moreover, the probes 3 and 5 exhibited good antiproliferative activity towards MCF-7 cells (IC50 = 8.5 μM, 10.3 μM) and a much lower cytotoxicity to normal cells compared with the positive control. It is expected that these novel fluorescent probes may provide useful tools for the theranostics of ER+ breast cancer.

A Self-Assembled "Albumin-Conjugate" Nanoprobe for Near Infrared Optical Imaging of Subcutaneous and Metastatic Tumors.

The need for in situ accurate identification of tumor assisted real-time image-guided surgical resection calls for new near-infrared fluorescence agents with high tumor-sensitivity and excellent biocompatibility. Here, an albumin-conjugate nanoparticle system HSA-Er-RI-Cl was designed, synthesized, and applied in cancer imaging, which simultaneously achieved the EPR effect, hypoxia-targeting, and EGFR-targeting property. Our novel nanoprobe is composed of human serum albumin (HSA) and double-targeting small molecule conjugate (Er-RI-Cl): a hypoxia-targeting heptamethine carbocyanine dye (RI-Cl) conjugated with a clinic anti-EGFR antagonist (Erlotinib) by covalent bonding. This conjugate could bind to albumin proteins, forming albumin-conjugate complexes, and those complexes self-assemble into particles with diameters of approximately 100 nm in the aqueous solution. The tumor hypoxia and EGFR targeting specificity of HSA-Er-RI-Cl was, respectively, evaluated in vitro and in vivo. Using murine xenograft subcutaneous and brain metastatic tumor models, we demonstrated that HSA-Er-RI-Cl is a highly potent tumor-targeting NIR agent for noninvasive imaging with remarkable tumor localization and excellent pharmacokinetic properties.

P4HB, a Novel Hypoxia Target Gene Related to Gastric Cancer Invasion and Metastasis.

Gastric cancer (GC) is a common tumor-associated lethal disease, and invasiveness and metastasis are primary challenges in its clinical treatment. Hypoxia microenvironment cannot be ignored in the process of metastasis. Hypoxia inducible factor-1α (HIF-1α) is the core component of the hypoxia signaling pathway. The aim of this study was to identify potential hub genes and signaling pathways associated with HIF-1α. We explored the invasiveness- and metastasis-associated phenotype of GC via bioinformatics analysis and molecular studies. Differentially expressed genes (DEGs) were identified in GC cells and HIF-1α-knockdown GC cells. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed, and a protein-protein interaction (PPI) network was constructed. Hub genes were identified via centrality analysis and Molecular Complex Detection (MCODE) module analysis. The findings suggested that prolyl 4-hydroxylase beta polypeptide (P4HB) has strong associations with HIF-1α. Further, we observed that HIF-1α and P4HB were upregulated in SGC-7901 and BGC-823 cells. In addition, inhibition of HIF-1α expression reduced invasion and metastasis in GC cells; this effect was partially reversed by P4HB overexpression. Our results confirm that P4HB plays a significant role in the regulatory network of HIF-1α. Therefore, HIF-1α and P4HB may be considered potential biomarkers of GC.

Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts.

Triple Negative Breast Cancer (TNBC) is aggressive, metastatic and drug-resistant, limiting the spectrum of effective therapeutic options for breast cancer patients. To date, anti-angiogenic agents have had limited success in the treatment of systemic breast cancer, possibly due to the exacerbation of tumor hypoxia and increased metastasis. Hypoxia drives increased expression of downstream effectors, including Carbonic Anhydrase IX (CAIX), a critical functional component of the pro-survival machinery required by hypoxic tumor cells. Here, we used the highly metastatic, CAIX-positive MDA-MB-231 LM2-4 orthotopic model of TNBC to investigate whether combinatorial targeting of CAIX and angiogenesis impacts tumor growth and metastasis in vivo to improve efficacy. The administration of a small molecule inhibitor of CAIX, SLC-0111, significantly reduced overall metastatic burden, whereas exposure to sunitinib increased hypoxia and CAIX expression in primary tumors, and failed to inhibit metastasis. The administration of SLC-0111 significantly decreased primary tumor vascular density and permeability, and reduced metastasis to the lung and liver. Furthermore, combining sunitinib and SLC-0111 significantly reduced both primary tumor growth and sunitinib-induced metastasis to the lung. Our findings suggest that targeting angiogenesis and hypoxia effectors in combination holds promise as a novel rational strategy for the effective treatment of patients with TNBC.

Benzotriazine Di-Oxide Prodrugs for Exploiting Hypoxia and Low Extracellular pH in Tumors.

Extracellular acidification is an important feature of tumor microenvironments but has yet to be successfully exploited in cancer therapy. The reversal of the pH gradient across the plasma membrane in cells that regulate intracellular pH (pHi) has potential to drive the selective uptake of weak acids at low extracellular pH (pHe). Here, we investigate the dual targeting of low pHe and hypoxia, another key feature of tumor microenvironments. We prepared eight bioreductive prodrugs based on the benzotriazine di-oxide (BTO) nucleus by appending alkanoic or aminoalkanoic acid sidechains. The BTO acids showed modest selectivity for both low pHe (pH 6.5 versus 7.4, ratios 2 to 5-fold) and anoxia (ratios 2 to 8-fold) in SiHa and FaDu cell cultures. Related neutral BTOs were not selective for acidosis, but had greater cytotoxic potency and hypoxic selectivity than the BTO acids. Investigation of the uptake and metabolism of representative BTO acids confirmed enhanced uptake at low pHe, but lower intracellular concentrations than expected for passive diffusion. Further, the modulation of intracellular reductase activity and competition by the cell-excluded electron acceptor WST-1 suggests that the majority of metabolic reductions of BTO acids occur at the cell surface, compromising the engagement of the resulting free radicals with intracellular targets. Thus, the present study provides support for designing bioreductive prodrugs that exploit pH-dependent partitioning, suggesting, however, that that the approach should be applied to prodrugs with obligate intracellular activation.

Targeting hypoxia downstream signaling protein, CAIX, for CAR T-cell therapy against glioblastoma.

Glioblastoma survival remains unchanged despite continuing therapeutic innovation. Herein, we aim to (i) develop chimeric antigen receptor (CAR) T cells with a specificity to a unique antigen, carbonic anhydrase IX (CAIX), which is expressed in the hypoxic microenvironment characteristic of glioblastoma, and (ii) demonstrate its efficacy with limited off-target effects. First we demonstrated expression of CAIX in patient-derived glioblastoma samples and available databases. CAR T cells were generated against CAIX and efficacy was assessed in 4 glioblastoma cell lines and 2 glioblastoma stem cell lines. Cytotoxicity of anti-CAIX CAR T cells was assessed via interferon gamma, tumor necrosis factor alpha, and interleukin-2 levels when co-cultured with tumor cells. Finally, we assessed efficacy of direct intratumoral injection of the anti-CAIX CAR T cells on an in vivo xenograft mouse model using the U251 luciferase cell line. Tumor infiltrating lymphocyte analyses were performed. We confirm that CAIX is highly expressed in glioblastoma from patients. We demonstrate that CAIX is a suitable target for CAR T-cell therapy using anti-CAIX CAR T cells against glioblastoma in vitro and in vivo. In our mouse model, a 20% cure rate was observed without detectable systemic effects. By establishing the specificity of CAIX under hypoxic conditions in glioblastoma and highlighting its efficacy as a target for CAR T-cell therapy, our data suggest that anti-CAIX CAR T may be a promising strategy to treat glioblastoma. Direct intratumoral injection increases anti-CAIX CAR T-cell potency while limiting its off-target effects.

Abstract 1230: Therapeutic targeting of hypoxia in experimental esophageal adenocarcinoma

Abstract Introduction: Esophageal adenocarcinoma (EAC) has become the predominant type of esophageal cancer in the United States, and prognosis of EAC remains poor despite modern combination therapies. Therefore, new therapeutic approaches are urgently needed. Hypoxia or insufficient tissue oxygenation contributes to EAC aggressiveness and poor clinical prognosis. Typically, EAC cell lines are used in two-dimensional (2D) cultures for screening of anticancer agents, and they do not represent the hypoxic tumor microenvironment. In this study, we established a novel three dimensional (3D) spheroid co-culture model of EAC. We then observed treatment response of chemo and hypoxia-targeting therapies in 2D, 3D and in vivo models of experimental EAC. Methods: EAC 3D spheroids were generated from co-culturing human EAC and fibroblast cell lines. NanoCulture® plates and dishes were used for 3D spheroid cultures. Hypoxic status was detected by adding hypoxia probe LOX-1 and fluorescent microscopy. Paclitaxel (PT), carboplatin (CP) and nanoparticle albumin-bound paclitaxel (NPT) were used as chemotherapeutic agents, whereas acriflavin was used as hypoxia-targeting agent. In vitro cell growth was detected by WST-1 assay, in vivo tumor growth was detected by measuring subcutaneous xenograft using OE19 EAC cells, apoptosis was detected by analysis of cleaved caspase 3 and PARP expressions, hypoxia-targeting was detected by analysis of HIF-1α expression and stem cell phenotype was detected by flow cytometric analysis of aldehyde dehydrogenase (ALDH) activity. Results: The results demonstrated that the 3D culture was more resistant to antiproliferative/proapoptotic effects of chemotherapeutic agents PT, CP, NPT and their combinations over 2D monolayer culture. Contrary to that, hypoxia-targeting agent acriflavin showed stronger antiproliferative/proapoptotic effects in 3D culture than in 2D culture. We observed strong expression of hypoxia inducible factor-1α (HIF-1α) in 3D culture with no expression of HIF-1α in 2D culture, and acriflavin treatment completely abolished the HIF-1α expression in 3D culture. We also observed hypoxia inside the 3D culture spheroids, but not in cells grown in 2D culture. Interestingly, we observed a reduced number of ALDH positive cells in 3D culture after acriflavin treatment compared to that after NPT treatment, indicating the preferential antiproliferative effect of acriflavin over NPT on cancer stem cells. In addition, acriflavin showed significant antitumor efficacy both as monotherapy and in combination with NPT. The xenograft tumor growth inhibition rate after a 2-week treatment with acriflavin, NPT and their combination was 44.97, 58.35 and 67.29 percent respectively (p<0.05). Conclusion: Thus 3D cultures may be better than 2D cultures in simulating the important in vivo tumor characteristic of hypoxia, and HIF-targeting therapy acriflavin could be a novel treatment strategy for EAC. Citation Format: Md Sazzad Hassan, Marisa Lenga, Lucia Petrova, Urs von Holzen. Therapeutic targeting of hypoxia in experimental esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1230.

Overcoming Chemoresistance in Advanced Ovarian Cancer Via Targeting Hypoxia

Overcoming Chemoresistance in Advanced Ovarian Cancer Via Targeting Hypoxia

Nanophotosensitizer-engineered Salmonella bacteria with hypoxia targeting and photothermal-assisted mutual bioaccumulation for solid tumor therapy

Bacteria-driven drug-delivery systems have attracted great attention for their enhanced therapeutic specificity and efficacy in cancer treatment. YB1, a particularly attractive genetically modified safe Salmonella Typhimurium strain, is known to penetrate hypoxic tumor cores with its self-driven properties while remarkably avoiding damage to normal tissues. Herein, nanophotosensitizers (indocyanine green (ICG)-loaded nanoparticles, INPs) were covalently attached to the surface of YB1 with amide bonds to develop a biotic/abiotic cross-linked system (YB1-INPs) for tumor precision therapy. YB1 microswimmer retained its viability after efficiently linking with INPs. This YB1-INPs treatment strategy demonstrated specific hypoxia targeting to solid tumors, perfect photothermal conversion, and efficient fluorescence (FL) imaging properties. Benefited from the combined contribution of tumor tissue destruction and the bacteria-attracting nutrients generation after photothermal treatment, the bioaccumulation of YB1-INPs was significantly improved 14-fold compared to no photothermal intervention. Furthermore, YB1-INPs pervaded throughout the large solid tumor (≥500 mm3). Under near-infrared (NIR) laser irradiation, YB1-INPs exhibited a dependable and highly efficient photothermal killing ability for eradicating the large solid tumor without relapse. This strategy of bacteria-driven hypoxia-targeting delivery has a great value for large solid tumors therapy with low toxicity and high efficiency.

Hypoxia promotes osteosarcoma cell proliferation and migration through enhancing platelet-derived growth factor-BB/platelet-derived growth factor receptor-β axis

Osteosarcoma is a primary malignant bone tumor, characterized by high therapeutic resistance and poor outcomes, due to unclear pathological mechanisms. It has been shown recently that the platelet-derived growth factor (PDGF)/platelet-derived growth factor receptor (PDGFR) pathway is closely associated with the pathogenesis of osteosarcoma. Hypoxia is a critical hallmark of tumor microenvironment that promotes the malignant phenotype in many solid tumors and a fundamental impediment to effective tumor therapy. In this study, we confirmed that hypoxia is an important feature of osteosarcoma, validated by the positive immunohistochemistry staining of hypoxia marker hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase IX (CAIX) in osteosarcoma tissue samples. More importantly, we discovered that hypoxia could transcriptionally upregulate the expression of both PDGF-BB and PDGFR-β in osteosarcoma cells in vitro. Likewise, we also established that hypoxia-induced PDGF-BB is strongly related to the enhanced cell proliferation and migration, by activating AKT, ERK1/2, and STAT3 signaling pathways. Notably, when using an antibody to block the autocrine of PDGF-BB, cell proliferation and migration were partially aborted in hypoxia. Collectively, we demonstrated that the hypoxia-activated PDGF-BB/PDGFR-β axis plays essential roles in osteosarcoma progression. These findings may shed light on the molecular pathogenesis of osteosarcoma, and provide a novel strategy for osteosarcoma treatment by combinational targeting hypoxia and PDGF-BB/PDGFR signaling.

Circulating miRNA Profiling of Women at High Risk for Ovarian Cancer

Survival of epithelial ovarian cancer patients remains poor without significant change over many decades. There is a need to better identify women at high risk (HR) for ovarian cancer. We propose that miRNA dysregulation may play critical roles in the early stages of ovarian cancer development. Circulating miRNAs may represent an important biomarker in this context, and miRNA profiling of serum in women at HR compared to those at low risk (LR) may give insights in tumor initiation pathways. There is also rationale for a specific focus on regulation of the androgen and its related hypoxia pathways in tumor initiation. We hypothesized that subsets of these pathway related miRNAs may be downregulated in the HR state. Serum from four HR and five LR women were sequenced and analyzed for 2083 miRNAs. We found 137 miRNAs dysregulated between the HR and LR groups, of which 36 miRNAs were overexpressed in HR and the vast majority (101 miRNAs or 74%) downregulated in the HR, when compared to LR serum. mRNA targets for the differentially expressed miRNAs were analyzed from three different miRNA-mRNA interaction resources. Functional association analysis of hypoxia and androgen pathway mRNA targets of dysregulated miRNAs in HR serum revealed that all but one of the miRNAs that target 52 hypoxia genes were downregulated in HR compared to LR serum. Androgen pathway analysis also had a similar expression pattern where all but one of the miRNAs that target these 135 identified genes were downregulated in HR serum. Overall, there were 91 differentially expressed miRNA-mRNA pairings in the hypoxia analysis. In the androgen-related analysis, overall, there were 429 differentially expressed miRNA-mRNA pairs. Our pilot study suggests that almost all miRNAs that are conserved and/or validated are downregulated in the HR compared to LR serum. This study, which requires validation, suggests that, via miRNA dysregulation, involvement of both hypoxia and its related androgen pathways may contribute to the HR state. This pilot study is the first report to our knowledge that studies circulating miRNA profiling of HR and LR women.

HIF-1 transcription activity: HIF1A driven response in normoxia and in hypoxia

BackgroundHIF1A (Hypoxia-Inducible-Factor 1A) expression in solid tumors is relevant to establish resistance to therapeutic approaches. The use of compounds direct against hypoxia signaling and HIF1A does not show clinical efficiency because of changeable oxygen concentrations in solid tumor areas. The identification of HIF1A targets expressed in both normoxia and hypoxia and of HIF1A/hypoxia signatures might meliorate the prognostic stratification and therapeutic successes in patients with high-risk solid tumors.MethodsIn this study, we conducted a combined analysis of RNA expression and DNA methylation of neuroblastoma cells silenced or unsilenced for HIF1A expression, grown in normoxia and hypoxia conditions.ResultsThe analysis of pathways highlights HIF-1 (heterodimeric transcription factor 1) activity in normoxia in metabolic process and HIF-1 activity in hypoxia in neuronal differentiation process. HIF1A driven transcriptional response in hypoxia depends on epigenetic control at DNA methylation status of gene regulatory regions. Furthermore, low oxygen levels generate HIF1A-dependent or HIF1A-independent signatures, able to stratify patients according to risk categories.ConclusionsThese findings may help to understand the molecular mechanisms by which low oxygen levels reshape gene signatures and provide new direction for hypoxia targeting in solid tumor.

MiR-193a-3p Promotes Radio-Resistance of Nasopharyngeal Cancer Cells by Targeting SRSF2 Gene and Hypoxia Signaling Pathway.

BackgroundRadio-resistance is an important barrier in nasopharyngeal carcinoma treatment. MicroRNAs are gene expression core regulators in various biological procedures containing cancer radio-resistance. Nevertheless, the clinical association between nasopharyngeal carcinoma and miR-193a-3p/SRSF2 remains unclear.Material/MethodsWe examined the miR-193a-3p level in radio-sensitive CNE-2 and radio-resistant CNE-1 NPC cell lines, and, based on a literature review, predicted SRSF2 to be the target gene of miR-193a-3p. We explored the expression of SRSF2 at protein and mRNA levels by transfecting either miR-193a-3p-mimic or antagomiR. Finally, we performed signaling pathway analysis to assess the possible role of miR-193a-3p/SRSF2 in signaling pathways.ResultsmiR-193a-3p promotes NPC radio-resistance, and the SRSF2 gene is the direct target for miR-193a-3p in NPC, and thus is negatively correlated with NPC radio-resistance. The hypoxia signaling pathway activity is strongly affected, and it is possible to use the downstream activity of the SRSF2 gene to show the effect of miR-193a-3p on radio-resistance in NPC cells.ConclusionsmiR-193a-3p mediates promotion of NPC radio-resistance.

Commentary on Some Recent Theses Relevant to Combating Aging: February 2019

Theses reviewed in this issue include Characterization and Modeling of Metabolic Stress Responses in Cellular Aging; Engineering Immunity: Enhancing T Cell Vaccines and Combination Immunotherapies for the Treatment of Cancer; Extracellular Inflammatory Signaling from Dysfunctional Telomeres; High-Throughput Microfluidic Labyrinth for the Label-Free Isolation of circulating tumor cells for Single-Cell Gene Expression Profiling; Oxygen Nanobubbles for Ultrasound-guided Targeting of Cancer Hypoxia; and The Eye as a Window to the Alzheimer's Disease Brain.

A novel "mosaic-type" nanoparticle for selective drug release targeting hypoxic cancer cells.

The surface potential of particles is a double-edged sword for nanomedicine. The negative charge can protect nanoparticles from clearance before they reach the tumor tissue; however, it is difficult to phagocytose the negative particles by target cells due to the negative potential of the cytomembrane. Preparing techniques to efficiently release the encapsulated drug from negative nanoparticles into target cells is a formidable challenge facing advanced drug delivery studies. Herein, we have developed a novel "mosaic-type" nanoparticle system (GA-Cy7-NP) for selective drug release targeting hypoxic cancer cells. In this system, hypoxia-targeting near-infrared dye (Cy7) moiety with a positive charge is conjugated to an antitumor agent, namely, gambogic acid (GA). This conjugate could self-assemble into nanoparticles with surfactin in an aqueous solution, where the Cy7 group is embedded in the negatively charged particle surface formed by surfactin. Most remarkably, the "mosaic-type" nanoparticles could selectively release the loaded drug conjugates into hypoxic cancer cells without particle internalization. Using in vitro PC3 cell and xenograft mouse models, we demonstrate that GA-Cy7-NP exhibits enhanced drug distribution in tumor cells and superior antitumor activity as compared to the prototype drug when evaluated in terms of cell proliferation, tumor growth, and angiogenesis assay.

Fucoidan-Manganese Dioxide Nanoparticles Potentiate Radiation Therapy by Co-Targeting Tumor Hypoxia and Angiogenesis.

Tumor hypoxia is a major mechanism of resistance to radiation therapy (RT), which is associated with poor prognosis in affected cancer patients. Various approaches to treat hypoxic and radioresistant cancers, including pancreatic cancer, have shown limited success. Fucoidan, a polysaccharide from brown seaweed, has antitumor and antiangiogenesis activities. Here, we discuss the development of fucoidan-coated manganese dioxide nanoparticles (Fuco-MnO2-NPs) and testing of the therapeutic potential with RT using pancreatic cancer models. In vitro data showed that Fuco-MnO2-NPs generated oxygen efficiently in the presence of H2O2 and substantially suppressed HIF-1 expression under a hypoxic condition in human pancreatic cancer cells. Fuco-MnO2-NPs reversed hypoxia-induced radioresistance by decreasing clonogenic survival and increasing DNA damage and apoptotic cell death in response to RT. In a BxPC3 xenograft mouse model, the combination treatment with Fuco-MnO2-NPs and RT resulted in a greater tumor growth delay than RT alone. Fucoidan-coated NPs, but not naked ones, further suppressed tumor angiogenesis, as judged by immunohistochemistry data with diminished expression of phosphorylated vascular endothelial growth factor receptor 2 (VEGFR2) and CD31. These data suggest that Fuco-MnO2-NPs may potentiate the effects of RT via dual targeting of tumor hypoxia and angiogenesis, and they are of great clinical potential in the treatment of hypoxic, radioresistant pancreatic cancer.

Integrin-α3 Is a New Marker of Long-Term Hematopoietic Stem Cells in Ex Vivo Expanded Cells

Hematopoietic stem cell (HSC) transplantation constitutes one of the most effective therapeutic strategies to treat numerous hematological diseases. Cord blood (CB) is one of the most attractive donor sources of stem cells for this procedure due to its rapid availability, HLA mismatches tolerance and low associated risk of chronic graft-versus-host disease. However, these advantages are offset by the limited cell dose in CB units, which can contribute to delayed hematopoietic engraftment following transplantation. Mastering ex vivo HSC expansion is therefore of great interest for clinical purposes and for genetic manipulation. HSCs can be functionally defined as either long-term (LT-HSC), providing life-long hematopoiesis and characterized by delayed engraftment pattern, or short-term repopulating stem cells (ST-HSC), providing early and transient hematopoietic recovery. Major hurdles hindering the study of these cell populations is the current inability to evaluate their content in cultured samples and the lack of understanding of the molecular mechanisms regulating stem cell self-renewal ex vivo. Those issues highly benefited from the discovery by our laboratory of the small molecule UM171, which promote HSC expansion ex vivo, as well as from the identification of EPCR as one of the most reliable surface markers for cultured HSCs.We now describe the identification of Integrin-α3 (ITGA3) as a novel marker for cultured HSCs. ITGA3 expression was found to be sufficient to split the primitive EPCR+CD90+CD133+CD34+CD45RA- HSC population in two functionally distinct fractions presenting only short-term (ITGA3-) and both short-term and long-term (ITGA3+) repopulating potential. ITGA3+ cells, as opposed to the ITGA3- fraction, exhibited robust multilineage differentiation potential and serial reconstitution ability in immunocompromised mice. This combination of markers identifies repopulating HSCs in culture by FACS beyond what is currently possible with other approaches, with a frequency of LT-HSC found in the ITGA3+ population estimated at 1:38 in day 7 UM171 expanded CB-cells. Moreover, lentiviral-mediated ITGA3 knockdown was shown to compromise the LT repopulating activity of cultured HSC in vivo. Gene expression profiling revealed striking molecular similarity between ITGA3+ and ITGA3- cells, showing overrepresentation of genes involved in fundamental hematopoietic programs known to govern HSC specification and function in both of these populations. However, ITGA3+ and ITGA3- subsets clearly clustered separately by principle component analysis, indicating broad differences in gene expression. Concordantly with their primitive phenotype, stem cell markers and cell quiescence are gene sets enriched in ITGA3+ cells, while progenitor markers, DNA replication, M/G1 and G2/M checkpoints, mRNA processing, reduction of hypoxia and Myc targets were significantly downregulated in these cells.Altogether, our results indicate that ITGA3 is a reliable marker for cultured HSCs, improving the accuracy of prospective HSC identification in culture. Deciphering the function of genes upregulated in primitive ITGA3+ HSCs will represent an invaluable resource for dissecting the genetic programs that govern hematopoietic stem cells biology. DisclosuresSauvageau:ExCellThera: Employment, Equity Ownership.

Synthesis and Evaluation of Multifunctional Fluorescent Inhibitors with Synergistic Interaction of Prostate-Specific Membrane Antigen and Hypoxia for Prostate Cancer.

Prostate cancer is one of the most common cancers in the world. It is widely known that prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer, and hypoxia is a common characteristic of many solid tumors, including prostate cancer. In this study, we designed multifunctional fluorescent inhibitors to target PSMA and tumor hypoxia in order to increase the tumor uptake of inhibitors. Novel PSMA inhibitors were prepared using lysine as the backbone to connect three different functional groups: the glutamate-urea-lysine (GUL) structure for inhibiting PSMA, 2-nitroimidazole for the hypoxia-sensitive moiety, and a near-infrared fluorophore (sulfo-Cyanine 5.5). According to the in vitro PSMA binding assay, novel fluorescent inhibitors were demonstrated to have nanomolar binding affinities. Multifunctional inhibitor 2 with one 2-nitroimidazole had a similar inhibitory activity to inhibitor 1 that did not contain the hypoxia targeting moiety, but multifunctional inhibitor 3 with two 2-nitroimidazoles showed lower inhibitory activity than inhibitor 1 due to the bulky structure of the hypoxia-sensitive group. However, in vivo optical imaging and ex vivo biodistribution studies indicated that both multifunctional inhibitors 2 and 3 had higher accumulation in tumors than inhibitor 1 due to a synergistic combination of PSMA and hypoxia targeting moieties. These observations suggest that this novel multifunctional strategy might be a promising approach to improve the diagnosis and therapy of prostate cancer.

Pathological Impacts of Chronic Hypoxia on Alzheimer's Disease.

Chronic hypoxia is considered as one of the important environmental factors contributing to the pathogenesis of Alzheimer's disease (AD). Many chronic hypoxia-causing comorbidities, such as obstructive sleep apnea syndrome (OSAS) and chronic obstructive pulmonary disease (COPD), have been reported to be closely associated with AD. Increasing evidence has documented that chronic hypoxia may affect many pathological aspects of AD including amyloid β (Aβ) metabolism, tau phosphorylation, autophagy, neuroinflammation, oxidative stress, endoplasmic reticulum (ER) stress, and mitochondrial and synaptic dysfunction, which may collectively result in neurodegeneration in the brain. In this Review, we briefly summarize the effects of chronic hypoxia on AD pathogenesis and discuss the underlying mechanisms. Since chronic hypoxia is common in the elderly and may contribute to the pathogenesis of AD, prospective prevention and treatment targeting hypoxia may be helpful to delay or alleviate AD.