Non-invasive bioluminescence imaging of caspase-3 activity in Breast Cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14557-e14557
Author(s):  
C. C. Olsen ◽  
F. Li ◽  
Z. He ◽  
W. Li ◽  
C. Li
Keyword(s):  
Breast Cancer ◽  
Drug Exposure ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Fold Increase ◽  
Reporter System ◽  
Reporter Protein ◽  
Reporter Proteins

e14557 Background: Apoptosis is a major form of tumor cells death during cytotoxic therapy. Understanding the kinetics of apoptosis would greatly facilitate development of more effective therapeutic approaches. In order to monitor apoptosis activities in vivo, we developed a novel bioluminescence-based reporter gene to detect caspase 3 activities, which are elevated at the execution phase of apoptosis. Methods: A caspase-3 reporter system was constructed by combining two different reporter proteins; green fluorescent protein (GFP) and firefly luciferase (FL) linked through multiple polyubiquitin domains with a caspase-3 recognition site. Under normal circumstances, the reporter proteins are rapidly degraded by the proteasome system.. During apoptosis, activated caspse 3 cleaves off the multi-ubiquitin domain from the reporter protein. This enable the GFP and luciferase fusion reporter to be stabilized and achieve a significant gain in GFP protein and luciferase activities, which in turn could be monitored both in vitro and in vivo. 4T1 cells transduced with CMV-luc or Caspase-3 reporter xenografts were treated with both chemotherapy and radiation therapy and monitored for apoptosis activity. Results: In vitro experiments demonstrated increased luciferase with increasing radiation dose reflective of apoptosis with background levels nearly undetectable. Taxol was associated with a time-dependent increase from 24 to 72hrs after drug exposure, indicating that apoptosis is a gradual, heterogeneous process. EGFP signal increased from 1.85% in controls to 80.6% in cells treated with 1uM Taxol. Xenografts showed nearly undetectable luciferase background with Cytoxan therapy resulting in a 90-fold increase, 10 Gy a 24 fold increase and fractionated RT (5Gy x3) with a 46-fold increase. Conclusions: We developed a novel in vivo caspase reporter based on the ubiquitous proteosome system of protein degradation and bioluminsecence imaging. This allowed us to assess activation of apoptosis in response to chemoradiation therapy in tissue culture and breast cancer xenografts over the course of 2–3 weeks, which has not been possible with other technologies. No significant financial relationships to disclose.

scholarly journals Quercetin Is a Flavonoid Breast Cancer Resistance Protein Inhibitor with an Impact on the Oral Pharmacokinetics of Sulfasalazine in Rats

Pharmaceutics ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 397
Author(s):  
Yoo-Kyung Song ◽  
Jin-Ha Yoon ◽  
Jong Kyu Woo ◽  
Ju-Hee Kang ◽  
Kyeong-Ryoon Lee ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Breast Cancer Resistance Protein ◽  
Fold Increase ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Cancer Resistance ◽  
Concentration Dependent Manner ◽  
Resistance Protein

The potential inhibitory effect of quercetin, a major plant flavonol, on breast cancer resistance protein (BCRP) activity was investigated in this study. The presence of quercetin significantly increased the cellular accumulation and associated cytotoxicity of the BCRP substrate mitoxantrone in human cervical cancer cells (HeLa cells) in a concentration-dependent manner. The transcellular efflux of prazosin, a stereotypical BCRP substrate, was also significantly reduced in the presence of quercetin in a bidirectional transport assay using human BCRP-overexpressing cells; further kinetic analysis revealed IC50 and Ki values of 4.22 and 3.91 μM, respectively. Moreover, pretreatment with 10 mg/kg quercetin in rats led to a 1.8-fold and 1.5-fold increase in the AUC8h (i.e., 44.5 ± 11.8 min∙μg/mL vs. 25.7 ± 9.98 min∙μg/mL, p < 0.05) and Cmax (i.e., 179 ± 23.0 ng/mL vs. 122 ± 23.2 ng/mL, p < 0.05) of orally administered sulfasalazine, respectively. Collectively, these results provide evidence that quercetin acts as an in vivo as well as in vitro inhibitor of BCRP. Considering the high dietary intake of quercetin as well as its consumption as a dietary supplement, issuing a caution regarding its food–drug interactions should be considered.

335 CYTOPLASMIC MICROINJECTION OF EXOGENOUS DNA IN IN VITRO AND IN VIVO DERIVED SHEEP EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 263
Author(s):  
F. Pereyra-Bonnet ◽  
A. Gibbons ◽  
M. Cueto ◽  
R. Bevacqua ◽  
L. Escobar ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Genetically Modified ◽  
Fold Increase ◽  
Egfp Expression ◽  
Control Group ◽  
Corpora Lutea ◽  
Exogenous Dna ◽  
Frozen Semen

Microinjection of DNA into the male pronucleus is a commonly used method to generate transgenic animals. However, it is only moderately efficient in several species because it requires proper male pronuclear visualisation, which occurs only in a narrow window of time in mice. The cytoplasmic microinjection of exogenous DNA (eDNA) is an alternative method that has not been fully investigated. Our objective was to evaluate if cytoplasmic microinjection of eDNA is capable of producing genetically modified embryos. In vitro and in vivo derived sheep embryos were cytoplasmically microinjected with pCX-EGFP previously incubated (5 min in a PVP droplet) with oolemma-cytoplasm fragments obtained from donor oocytes by microsurgery. A control group using microinjected plasmid alone was included in the in vivo procedure. For in vitro microinjection, IVF embryos were microinjected with circular plasmid with promoter (50 or 500 ng μL–1) or without promoter (50 ng μL–1) at 6 h after fertilization. The IVF was performed following (Brackett and Olliphant 1975 Biol. Reprod. 12, 260–274) with 15 × 106 spermatozoa mL–1, and presumptive zygotes were cultured in SOF. The expression of enhance green fluorescent protein (EGFP) was determined under blue light. For in vivo microinjection, embryos from superovulated sheep (by standard procedures) were recovered and microinjected with 50 ng μL–1 of linearized plasmid without promoter at 12 h after laparoscopic insemination with frozen semen (100 × 106 spermatozoa per sheep). Plasmid without promoter was used to avoid any possible cytotoxic effect produced by EGFP expression. The microinjection of IVF embryos with 50 ng μL–1 of plasmid was the best condition to produce embryos expressing eDNA (n = 96; 46.9% cleaved; 12.2% blastocysts; 53.0 and 4.1% of green embryos and blastocysts, respectively). Variables between the groups with or without promoter IVF were not statistically different (Fisher test: P < 0.05); however, when 500 ng μL–1 was microinjected, no blastocysts were obtained. In the in vivo embryo production group, 111 presumptive zygotes were microinjected (n = 37; with plasmid alone) from 16 donor sheep (11.5 ± 4.0 corpora lutea; 8.4 ± 4.8 presumptive zygotes recovered; 74.3% recovery rate). The mean time from injection to cleavage was 18.0 ± 4.5 h, and the percentage of cleavage and damage (due to the embryo injection) were >70% and <10%, respectively. Fifty-eight good quality embryos were transferred into the oviducts of 19 surrogate ewes; 12 of them are pregnant (63.1%). The presence of green IVF embryos demonstrates that eDNA was transported to the nucleus after cytoplasmic injection. We believe that the multi-fold increase (50- to 100-fold) in plasmid concentration compared with that used by others was the key step to our successful cytoplasmic microinjection. Accordingly, the new/old methodology described in this study provides an easy DNA construct delivery system of interest for the implementation of early reprogramming events. In addition, results obtained in the near future using in vivo cytoplasmic microinjection with high concentrations of eDNA could revalidate this technique for producing genetically modified large animals.

scholarly journals In Vitro and In Vivo Anticancer Activity of the Most Cytotoxic Fraction of Pistachio Hull Extract in Breast Cancer

Molecules ◽  
2020 ◽  
Vol 25 (8) ◽  
pp. 1776 ◽  
Author(s):  
Maryam Seifaddinipour ◽  
Reyhaneh Farghadani ◽  
Farideh Namvar ◽  
Jamaludin Bin Mohamad ◽  
Nur Airina Muhamad
Keyword(s):  
Breast Cancer ◽  
Caspase 3 ◽  
Active Agent ◽  
Suppressive Effect ◽  
Caspase 8 ◽  
Nuclear Staining ◽  
Active Components ◽  
Mcf 7

Pistacia (Pistacia vera) hulls (PV) is a health product that has been determined to contain bioactive phytochemicals which have fundamental importance for biomedical use. In this study, PV ethyl acetate extraction (PV-EA) fractions were evaluated with the use of an MTT assay to find the most cytotoxic fraction, which was found to be F13b1/PV-EA. After that, HPTLC was used for identify the most active compounds. The antioxidant activity was analyzed with DPPH and ABTS tests. Apoptosis induction in MCF-7 cells by F13b1/PV-EA was validated via flow cytometry analysis and a distinctive nuclear staining method. The representation of genes like Caspase 3, Caspase 8, Bax, Bcl-2, CAT and SOD was assessed via a reverse transcription (RT_PCR) method. Inhabitation of Tubo breast cancer cell development was examined in the BALB-neuT mouse with histopathology observations. The most abundant active components available in our extract were gallic acid and the flavonoid quercetin. The F13b1/PV-EA has antiradical activity evidence by its inhibition of ABTS and DPPH free radicals. F13b1/PV-EA displayed against MCF-7 a suppressive effect with an IC50 value of 15.2 ± 1.35 µg/mL. Also, the expression of CAT, SOD, Caspase 3, Caspase 8 and Bax increased and the expression of Bcl-2 decreased. F13b1/PV-EA dose-dependently inhibited tumor development in cancer-induced mice. Thus, this finding introduces F13b1/PV-EA as an effectual apoptosis and antitumor active agent against breast cancer.

scholarly journals Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A

2011 ◽  
Vol 300 (5) ◽  
pp. C989-C997 ◽  
Author(s):  
Pimthanya Wanichawan ◽  
William E. Louch ◽  
Kristin H. Hortemo ◽  
Bjørg Austbø ◽  
Per Kristian Lunde ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Phosphorylation Site ◽  
Full Length ◽  
Kinase A

The cardiac Na+/Ca2+ exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-32P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.

Role of IFN-γ and IL-2 in rat lung epithelial cell migration and apoptosis after oxidant injury

2004 ◽  
Vol 286 (1) ◽  
pp. L4-L14 ◽  
Author(s):  
Olivier Lesur ◽  
Marcel Brisebois ◽  
Alexandre Thibodeau ◽  
Frédéric Chagnon ◽  
Denis Lane ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Primary Cultures ◽  
Fold Increase ◽  
Protective Role ◽  
Scatchard Analysis ◽  
Membrane Expression ◽  
Extracellular Signal ◽  
Ifn Γ

In the present study, IFN-γ exposure to primary cultures of rat type II epithelial cells (TIIP) upregulated membrane expression of the common γ-chain of the IL-2 receptor (∼2.5- to 4-fold increase) and redistributed receptor affinity in TIIP, as assessed by Western blot, cell, and tissue histochemistry and Scatchard analysis. As for restitution processes of the lung epithelium, functionality of IL-2R on TIIP was conditional to IFN-γ exposure: 1) IFN-γ priming promoted a fivefold increase of IL-2-driven TIIP locomotion ( P < 0.05 vs. control at 100 U/ml) and 2) IFN-γ coincubation with IL-2 reduced bleomycin-induced TIIP apoptosis in vitro by 25% (caspase-3 activity) and by ∼70% (TdT-mediated dUTP nick end labeling/4′,6′-diamidino-2-phenylindole assay) as well as in vivo by ∼90% (caspase-3 activity; P < 0.05 vs. control). Sustained p42/44 extracellular signal-regulated kinase activity played a protective role in this process, whereas specific inhibition by PD-98059 (50 μM) significantly reversed bleomycin-induced TIIP apoptosis ( P < 0.05 vs. control). From these in vitro and in vivo data, it is proposed that combinations of IFN-γ and IL-2 can drive repair activity of TIIP by stimulating migration and preventing programmed cell death, both of which are speculated to be very fast restitution events after oxidant-induced acute lung injury.

Enriched MicroRNA-126 Bioactivity Marks the Primitive Compartment In AML and Regulates LSC Numbers

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 94-94
Author(s):  
Eric Lechman ◽  
Bernhard Gentner ◽  
Hidefumi Hiramatsu ◽  
Kristin J Hope ◽  
Katsuto Takenaka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Colony Formation ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Biologically Active ◽  
Reporter System ◽  
Differentiation Marker

Abstract Abstract 94 Previous work has shown miRNAs to be dysregulated in acute myeloid leukemia (AML), however, there is little known regarding miRNA expression and function in leukemia stem cells (LSC). In order to elucidate the role of miRNA in LSC, we performed miRNA profiling on fractionated subpopulations of primary AML patient samples. Supervised analysis guided by the in vivo SCID leukemia initiating capacity (SL-IC) of each sub-population generated a unique miRNA signature associated with LSC enriched fractions. An in vitro antagomir-based functional miRNA knockdown screen identified miR-126, our top array candidate, for further study. After RT-PCR validation, the biological activity of miR-126 was confirmed at single cell resolution by using a novel bidirectional lentivirus miRNA reporter system in the 8227 cell line in vitro and within primary AML patient samples xenografted into immune-deficient NSG mice. These data suggest that primitive AML cells may express high levels of bioactive miR-126 relative to more “differentiated” blast populations. To test the hypothesis that AML stem cells are marked by high miR-126 bioactivity, we FACS sorted miR-126 genetic reporter vector transduced primary AML patient samples and transplanted these populations into immune-compromised secondary mouse recipients. The results of these proof-of-concept experiments demonstrates our ability to prospectively isolate LSC enriched fractions in all 4 AML patient samples tested using only a single biomarker, miR-126. Finally, to understand the functional relevance of miR-126 expression within primitive AML cells, stable enforced expression and knockdown of miR-126 was achieved using lentiviral vectors. Enforced expression of miR-126 in CD34+CD38- 8227 cells resulted in reduced AML blast colony formation, an increase/maintenance of CD34+ cells and a decrease in differentiation marker positive (CD14, CD15) AML blasts. Similarly, enforced miR-126 expression in 4 primary AML xenografts resulted in a several fold increase of CD34+CD117+ lentivirus marked leukemia cells after 12 weeks. In addition, the miR-126 cells showed reduced differentiation marker expression (CD14, CD15) with no significant differences in AML graft size. To determine if the expanded population had SL-IC activity or was a downstream leukemic progenitor, limiting dilution assays were performed by transplantation of FACS sorted lentivirus marked cells into secondary recipient mice for 12 weeks. A 13 fold increase in LSC activity was observed with miR-126 forced expression compared to control vector expressing cells. These data suggest that high levels of miR-126 bioactivity may support self-renewal/maintenance of primitive AML cells at the cost of aberrant differentiation. Conversely, in vitro knockdown of miR-126 in CD34+CD38- 8227 cells increased AML blast colony formation, while no phenotype was observed in xenotransplanted primary AML, with secondary LDA transplant experiments ongoing. Target prediction algorithms and previously described target genes were used to ascertain the principal signaling pathway(s) under direct control of miR-126 in primitive AML cells. In summary, these experiments demonstrate that miR-126 is more abundantly expressed and biologically active within the leukemia stem/progenitor cell compartment of the AML functional hierarchy and serves to regulate AML stem cell numbers. Disclosures: No relevant conflicts of interest to declare.

Response and Resistance to Bromodomain Inhibition in AML Driven By Hyperactive Ras Signaling

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1654-1654
Author(s):  
Benjamin Huang ◽  
Eugene Hwang ◽  
Anica M. Wandler ◽  
Ben Powell ◽  
Gideon Bollag ◽  
...  
Keyword(s):  
Drug Exposure ◽  
Therapeutic Potential ◽  
Clonal Evolution ◽  
Fold Increase ◽  
Pathological Examination ◽  
Pharmacokinetic Analysis ◽  
Ras Signaling ◽  
Hematopoietic Stem

Abstract Background: Acute myeloid leukemia (AML) is an aggressive hematologic cancer characterized by clonal proliferation of hematopoietic stem and progenitor cells that exhibit impaired differentiation. Event free survival for patients with AML remains poor despite intensive myelosuppressive therapies and improvements in supportive care measures. This underscores the need for novel, biologically based therapies. Somatic mutations that deregulate epigenetic programs (e.g. DNMT3A, TET2, IDH1/2, EZH2, ASXL1) and signal transduction pathways (e.g., FLT3, NRAS, KRAS) frequently coexist in AML. While the former class of mutations is hypothesized to promote a chromatin state that is permissive for AML development and essential for leukemia maintenance, experimental data also suggest that signaling mutations play a central role in driving leukemic growth in vivo. Thus, simultaneously targeting the abnormal epigenetic landscape and aberrant signaling pathways in AML is a rational new therapeutic approach. Recent publications identified the bromodomain and extraterminal (BET) proteins, an important class of epigenetic reader proteins, as particularly promising therapeutic targets in AML. While these studies support the therapeutic potential of BET inhibition in AML, they have limitations. These include their dependence on exogenous overexpression of oncogenes, failure to inform potential combination therapeutic strategies, and a reliance on monoclonal in vitro systems that do not recapitulate the inherent genetic heterogeneity of human cancers. Methods: We previously generated a heterogeneous collection of murine AMLs by infecting Nras, Kras, and Nf1 mutant mice with the MOL4070 retrovirus, which exhibit distinct retroviral integrations that are maintained upon transplantation into sublethally irradiated recipient mice. We first established 15 mg/kg/day as the maximally tolerated dose of PLX51107, a selective and potent BET inhibitor, in sublethally irradiated mice in a C57Bl/6 x 129sv/J strain background. We performed pharmacokinetic analysis, which demonstrated excellent drug exposure at doses of 10 and 15 mg/kg/day. We next treated cohorts of recipient mice with PLX51107 (10 mg/kg/day) and in combination with the MEK inhibitor PD0325901 (PD901; 1.5 mg/day). Mice that appeared ill were euthanized and underwent full pathological examination. Despite continuous drug treatment, all recipient mice eventually succumbed to progressive AML. Results: We enrolled eight AMLs, including four with a Nras(G12D) mutation, two with a Kras(G12D) mutation, and two with Nf1 inactivation. Recipient mice received 450 cGy of sublethal irradiation followed by 2x10E6 leukemia cells via tail vein injection. Recipient mice were randomized to receive vehicle (n = 4 for each AML), PLX51107 (n = 5), or PLX51107+PD901 (n = 5). PLX51107 markedly extended the survival of recipients transplanted with Nras(G12D) AMLs 6695, 6606, and 6613 that was further enhanced by PD901 (Fig. 1A). Whereas, PD901 resulted in a 1.5-fold increase in survival over vehicle-treated mice, PLX51107 alone resulted in a 4-fold increase in survival and PLX51107+PD901 in a nearly 6-fold increase in survival in this cohort of Nras(G12D) AMLs. Surprisingly, the response to PLX51107 was blunted in Kras(G12D) and Nf1 inactivated AMLs compared to Nras(G12D) AMLs. The observation of novel MOL4070 integration sites in relapsed AMLs provided definitive evidence of clonal evolution (Fig. 1B). Importantly, we went on to show that drug-treated clones emerging at relapse demonstrate intrinsic drug resistance by re-transplanting these leukemias into secondary recipients and re-treating them in vivo (Fig. 1C). Conclusion: PLX51107 shows impressive efficacy in a panel of primary AMLs treated in vivo that is further enhanced by PD901. The differential response between Nras(G12D) and Kras(G12D)/Nf(-/-) AMLs leads to the intriguing and unexpected hypothesis that the type of hyperactive Ras signaling mutation may influence the response to BET inhibition in AML. We are interrogating relapsed AMLs to identify and functionally validate candidate mechanisms underlying drug response and resistance through the use of established strategies to directly compare untreated and relapsed leukemias. Ongoing studies include assessing retroviral integrations and performing Western blotting, whole exome sequencing, RNA-seq, and ChIP-seq. Disclosures Powell: Plexxikon: Employment. Bollag:Plexxikon Inc.: Employment.

scholarly journals Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins

2007 ◽  
Vol 189 (18) ◽  
pp. 6564-6571 ◽  
Author(s):  
Jennifer S. Choy ◽  
Latt Latt Aung ◽  
A. Wali Karzai
Keyword(s):  
Messenger Rna ◽  
Fluorescent Protein ◽  
Protein Product ◽  
Lon Protease ◽  
Reporter Protein ◽  
Direct Role ◽  
Mutant Cells ◽  
Protein Tagging

ABSTRACT Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with λ-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins.

scholarly journals TMIC-59. INVESTIGATING PHYSIOLOGY OF THERAPY RESISTANT, TUMOR-INITIATING GBM CELLS IN HYPOXIC NICHES USING SPATIALLY-SENSITIVE HYPOXIA REPORTER SYSTEM AND SINGLE-CELL RNA SEQUENCING

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi260-vi261
Author(s):  
Anirudh Sattiraju ◽  
Valerie Marallano ◽  
Roland Friedel ◽  
Hongyan Zou
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Reporter System ◽  
T Cell Infiltration ◽  
Single Cell Rna Sequencing ◽  
Stem Cell Lines

Abstract Glioblastoma (GBM) is the most common and lethal brain cancer that invariably recurs after therapy due to presence of resistant GBM cells within hypoxic and peri-necrotic regions. Eradicating such GBM cells, which constitute a major source of tumor recurrence, is important to curb disease relapse. An endogenously expressed, spatially sensitive hypoxia reporter would therefore be a valuable tool to evaluate hypoxic zones in GBM in detail, and to measure the efficacy of hypoxia-activated drugs. For this purpose, we engineered a lentiviral vector that carries a hypoxia reporter, consisting of HIF response elements (HRE) that drive expression of UnaG fluorescent protein, which fluoresces independent of oxidative maturation. We validated the sensitivity of our reporter in vitro using U87MG, GBM2, and patient-derived GBM stem cell lines, and we performed intracranial transplantations of GBM cells in SCID mice to identify cells undergoing hypoxic stress in in vivo microenvironment. In addition, GL261 murine GBM cells with hypoxia reporter were intracranially implanted in C57BL/6 mice as syngeneic model for studies on immune responses. Brains from our transplant studies were dissociated and single-cell RNA sequencing (Drop-Seq) was performed to investigate heterogeneity in response to hypoxia within GBM cells and the cellular composition of microenvironment. We will also apply a hypoxia-activated prodrug, Evofosfamide (Evo), in our ongoing studies that can potentially eradicate hypoxic tumor cells and increase T cell infiltration and reverse immune suppression. As hypoxic niches are thought to confer resistance to radiation therapy (XRT), combining XRT with Evo could thus improve therapy efficacy. Our hypoxia gene reporter, combined with single-cell transcriptomics, could therefore serve as an effective tool to enable fundamental investigation of GBM microenvironment and could be used to evaluate therapies targeting tumor microenvironment to enhance GBM patient survival.

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