Fast Quantitation of TGF-β Signaling Using Adenoviral Reporter

Teixeira, Adilson Fonseca; Iaria, Josie; Zhu, Hong-Jian

doi:10.1007/978-1-0716-2277-3_2

Cited by 5 publications

(5 citation statements)

References 18 publications

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“…To evaluate if the amplification of TGF-β signaling induced by sEVs in vitro could be reproduced in vivo, a breast cancer xenograft mouse model 39 , 40 was used. Unlabeled MDA231 cells and Gaussia luciferase-labeled MDA231 (MDA.Gluc) cells were orthotopically and contralaterally implanted and investigated in vivo as breast cancer cells highly responsive to TGF-β (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…TGF-β activity in EVs was quantified by dual luciferase assay in MDA-MB-231 cells infected with Ad-CAGA-FLuc and Ad-CMV-GLuc adenoviruses as previously described. 39 To establish a standard curve, the TGF-β/SMAD3 signaling reporter (Ad-CAGA-Fluc) activity was determined in cell cultures treated with rhTGF-β1 at increasing concentrations. Simultaneously, the TGF-β/SMAD3 signaling reporter (Ad-CAGA-FLuc) activity was quantified in cell cultures treated with EVs and EV-depleted conditioned medium at increasing volumes.…”

Section: Methodsmentioning

confidence: 99%

“…To establish a model of metastatic breast cancers highly responsive to TGF-β, NOD-SCID mice were orthotopically and contralaterally implanted with MDA231 cells (1× MDA231 tumor/mouse; right mammary fat pad) and Gaussia luciferase-labeled MDA231 (MDA.Gluc) cells (1× MDA.Gluc tumor/mouse; left mammary fat pad). To quantify the TGF-β/SMAD3 signaling activity in primary tumors, 39 , 40 MDA.Gluc tumors infected with TGF-β/SMAD3 signaling reporter (Ad-CAGA-Fluc) were injected with 20 µL solution containing 5 µg MDA231-sEVs (2 ng TGF-β activity) or vehicle (PBS 1×). Intratumoral injections were administered twice weekly (dpi 17-27) until in vivo quantification of the TGF-β/SMAD3 signaling activity in MDA.Gluc tumors.…”

Section: Methodsmentioning

confidence: 99%

“…The bioluminescence signal emitted by the fat pad area was quantified using an In Vivo Imaging System (IVIS 200 Series, Caliper Life Sciences). 39 , 40 Mice lying supine were imaged each 1–2 min for 30 min following intraperitoneal injection with 150 mg/kg VivoGlo™ Luciferin, In Vivo Grade (P1043, Promega Corporation Australia). To image different areas of target tumors aiming at establishing the maximum TGF-β/SMAD3 signaling activity, mice were tilted on their flank after the emitted bioluminescence reached a plateau.…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis

Teixeira,

Wang,

Iaria

et al. 2023

Sig Transduct Target Ther

Self Cite

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Metastasis is the leading cause of cancer-related deaths. Transforming growth factor beta (TGF-β) signaling drives metastasis and is strongly enhanced during cancer progression. Yet, the use of on-target TGF-β signaling inhibitors in the treatment of cancer patients remains unsuccessful, highlighting a gap in the understanding of TGF-β biology that limits the establishment of efficient anti-metastatic therapies. Here, we show that TGF-β signaling hyperactivation in breast cancer cells is required for metastasis and relies on increased small extracellular vesicle (sEV) secretion. Demonstrating sEV’s unique role, TGF-β signaling levels induced by sEVs exceed the activity of matching concentrations of soluble ligand TGF-β. Further, genetic disruption of sEV secretion in highly-metastatic breast cancer cells impairs cancer cell aggressiveness by reducing TGF-β signaling to nearly-normal levels. Otherwise, TGF-β signaling activity in non-invasive breast cancer cells is inherently low, but can be amplified by sEVs, enabling invasion and metastasis of poorly-metastatic breast cancer cells. Underscoring the translational potential of inhibiting sEV trafficking in advanced breast cancers, treatment with dimethyl amiloride (DMA) decreases sEV secretion, TGF-β signaling activity, and breast cancer progression in vivo. Targeting both the sEV trafficking and TGF-β signaling by combining DMA and SB431542 at suboptimal doses potentiated this effect, normalizing the TGF-β signaling in primary tumors to potently reduce circulating tumor cells, metastasis, and tumor self-seeding. Collectively, this study establishes sEVs as critical elements in TGF-β biology, demonstrating the feasibility of inhibiting sEV trafficking as a new therapeutic approach to impair metastasis by normalizing TGF-β signaling levels in breast cancer cells.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis

Teixeira,

Wang,

Iaria

et al. 2023

Sig Transduct Target Ther

Self Cite

View full text Add to dashboard Cite

show abstract

“…Accordingly, these methods are typically associated with reduced sensitivity and specificity. Thus, overcoming these limitations to improve patient diagnosis and monitoring will require the establishment of sensitive methods with a focus on detecting and tracking alterations in biomarkers with real impact on cancer progression, especially EMT drivers [147][148][149].…”

Section: Future Directionsmentioning

confidence: 99%

A New Era of Integration between Multiomics and Spatio-Temporal Analysis for the Translation of EMT towards Clinical Applications in Cancer

Fonseca Teixeira,

Wu,

Luwor

et al. 2023

Cells

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Epithelial-mesenchymal transition (EMT) is crucial to metastasis by increasing cancer cell migration and invasion. At the cellular level, EMT-related morphological and functional changes are well established. At the molecular level, critical signaling pathways able to drive EMT have been described. Yet, the translation of EMT into efficient diagnostic methods and anti-metastatic therapies is still missing. This highlights a gap in our understanding of the precise mechanisms governing EMT. Here, we discuss evidence suggesting that overcoming this limitation requires the integration of multiple omics, a hitherto neglected strategy in the EMT field. More specifically, this work summarizes results that were independently obtained through epigenomics/transcriptomics while comprehensively reviewing the achievements of proteomics in cancer research. Additionally, we prospect gains to be obtained by applying spatio-temporal multiomics in the investigation of EMT-driven metastasis. Along with the development of more sensitive technologies, the integration of currently available omics, and a look at dynamic alterations that regulate EMT at the subcellular level will lead to a deeper understanding of this process. Further, considering the significance of EMT to cancer progression, this integrative strategy may enable the development of new and improved biomarkers and therapeutics capable of increasing the survival and quality of life of cancer patients.

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Systemic brain dissemination of glioblastoma requires transdifferentiation into endothelial-like cells via TGF-β-ALK1-Smad1/5 signaling

Ware,

Teixeira,

Iaria

et al. 2025

Neoplasia

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Fast Quantitation of TGF-β Signaling Using Adenoviral Reporter

Cited by 5 publications

References 18 publications

Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis

Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis

A New Era of Integration between Multiomics and Spatio-Temporal Analysis for the Translation of EMT towards Clinical Applications in Cancer

Systemic brain dissemination of glioblastoma requires transdifferentiation into endothelial-like cells via TGF-β-ALK1-Smad1/5 signaling

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