EZ4U Cell Proliferation & Cytotoxicity Assay
Report on novel prostate tumor cell targets
The Biomedica EZ4U – Cell Proliferation & Cytotoxicity Kit was cited in a high tier journal studying novel prostate tumor endothelial cell targets. The study highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis:
EZ4U Cell Proliferation & Cytotoxicity Assay
- Use of non-radioactive & non-toxic substances
- Easy one-step procedure on living cells
- Cited in more than 225 publications
More information can be found in our brochure: EZ4U – cat. no. BI-5000
Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer.
Heidegger I, Fotakis G, Offermann A, Goveia J, Daum S, Salcher S, Noureen A, Timmer-Bosscha H, Schäfer G, Walenkamp A, Perner S, Beatovic A, Moisse M, Plattner C, Krogsdam A, Haybaeck J, Sopper S, Thaler S, Keller MA, Klocker H, Trajanoski Z, Wolf D, Pircher A. Mol Cancer. 2022 Jun 18;21(1):132. doi: 10.1186/s12943-022-01597-7. PMID: 35717322; PMCID: PMC9206324. Full text
Background: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME.
Methods: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture experiments as well as a PCa xenograft mouse model.
Results: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro.
Conclusions: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.
Procedure of the EZ4U colorimetric test
The cell proliferation Assay kit EZ4U is non-toxic and non-radioactive that is based on the reduction of tetrazolium salt to colored formazan in living cells. Thus, the assay discriminates between living and dead cells since the process requires functional mitochondria. The assay is very easy to perform – add EZ4U reagent directly to the cells cultured, incubate and detect absorbance of the supernatant by a reader. The assay has been applied in numerous cells as reported in more than 225 publications e.g.
Fasting improves therapeutic response in hepatocellular carcinoma through p53-dependent metabolic synergism. Krstic J et al., Sci Adv. 2022. 21;8(3):eabh2635.
“Cell viability was analyzed using EZ4U assay (Biomedica Immunoassays) according to the manufacturer’s instructions. Briefly, at the end of treatment, the medium was replaced with fresh GM (200 μl per well) and 20 μl per well of EZ4U working solution. After 2 hours of incubation at 37°C, the absorbance was measured at 492 nm with a reference wavelength of 620 nm (Spark 10M multimode microplate reader).”
Syndecan-4 Is a Key Facilitator of the SARS-CoV-2 Delta Variant’s Superior Transmission. Hudák A et al., Int J Mol Sci. 2022. 12;23(2):796.
“Cell Viability Measurements: The effect of the applied spike proteins on cell viability was assessed with the EZ4U cell proliferation assay (Biomedica Gmbh, Vienna, Austria, cat. no. BI-5000), according to the instructions of the manufacturer. Absorbance was measured with a BioTek Cytation 3 multimode microplate reader.”
- Water-soluble tetrazolium compound penetrates cell membrane
- In mitochondria reduction takes place and results in intense colored formazan which is water-soluble
- The reagent Formazan is secreted into culture medium and measured with an ELISA reader at 450 nm or 492 nm
- Sample type – culture medium
- Sample size – 200 µl / test
- Detection limit – depends on respective cell line. Some xamples are shown in the product insert
- Incubation time – between 2-5 hours
What does the EZ4U kit include?
The reagents supplied per kit are sufficient for testing 10 x 96 well microtiter plates and contain:
- Substrate – lyophilized 10 vials
- Activator solution – ready to use 1 x 30 ml
Examples of cell types used in the EZ4U assay
– Endothelial cells (primary isolated from biopsies or cell-lines)
– Peripheral blood mononuclear cells (PBMCsec)
– HeLa (human cervical cancer) and HEK293A (human embryonic kidney) cell lines
– Human aortic smooth muscle cells (HAoSMCs)
– Huh7 (human liver carcinoma, CLS Cell Lines)
– Colon carcinoma cell models SW480
– CLC CTC cell lines (BHGc7, BHGc10, BHGc16, BHGc26 and UHGc5)
– CRC cell line HT29, DLD-1, HCT116,primary CRC cell line CG08
– HepG2 and Huh6 clone 5 cell lines
– Primary human skin cells – keratinocytes and fibroblasts
– Bovine mammary epithelial cells (MAC-T)
– Human primary chondrocytes
– HL-60 leukemia cells
– A172 and T98G glioblastoma cells
– Clonal preosteoblastic cell line MC3T3-E1-derived from newborn mouse calvaria
– Osteocyte-like MLO-Y4 cell line
– Preosteoclastic, macrophage-like RAW 264.7 cell line
– Primary HUVECs – human vascular endothelial cells
– MCF-7, T47D and MDA-MB-231 breast cancer cell lines
and many more..