ProSense on the ASK


Overview


Traditional mouse models of cancer rely primarily on ex vivo measurements of disease morphology and histologic analysis for the assessment of tumors. These measurements of disease may be distant from the actual biological targets of interest and can be time consuming, expensive, and impractical. By using NIR (Near-Infrared) in vivo imaging probes in combination with FMT, the biological processes that change with disease progression and therapeutic response can be visualized non-invasively over time.

ProSense™ agents were developed to specifically look at the expression and activity of key disease associated proteases. ProSense agents are optically silent until in the presence of these proteases and can be used to easily monitor the activity of these proteases in real time. PerkinElmer offers three different ProSense activatable agents. ProSense 680 is activated by Cathepsin B, L, S and Plasmin. ProSense 750 FAST is activated by Cathepsin B, L, S, K, V and D. ProSense 750 EX is activated by Cathepsin B, L, S and Plasmin.  

prosensefig1.jpg
Figure 1: Mechanism of ProSense activation. The ProSense probes are comprised of a pair of quenched fluorophore separated by a cleavable linker. Upon cleavage of the linker by the specific disease-related proteases, the probe becomes highly fluorescent. Therefore, the fluorescent signal is directly proportional to the expression and activity levels of the proteases.


Products and catalog numbers


ProductCatalog NumberEx/Em wavelength (nm)Molecular weight (g/mol)Validated ExperimentsApplicationsStorage and Stability
ProSense 680NEV10003680/700400,000In vivo/Ex vivo
Flow cytometry
In vitro microscopy
Oncology
Arthritis
Technical Data Sheet
ProSense 750 FASTNEV11171750/780400,000In vivo/Ex vivo
Flow cytometry
In vitro microscopy
Oncology
Arthritis
Technical Data Sheet
ProSense 750EXNEV10001EX750/770450,000In vivo/Ex vivo
Flow cytometry
In vitro microscopy
Oncology
Inflammation
Technical Data Sheet


Using ProSense in vivo


The recommended procedure for in vivo imaging with ProSense agents is administration via tail vein injection and imaging 24 hours post injection.
  • Imaging in Arthritis: ProSense agents can be used as a marker for disease progression and therapeutic response in animal models of arthritis.
  • Imaging in Oncology: ProSense agents can be used as a marker for disease progression in animal tumor models.
  • Pulminary Disease: ProSense agents to assess cathepsin activity in activated neutrophils.
ProSenseRoute of InjectionMouse Dose (25 g)Rat Dose (250 g)Blood t 1/2Tissue t 1/2Optimal imaging timeOptimal Re-injection Time (complete clearance)Route of Metabolism/ background tissueFMT and IVIS settings
ProSense 680IV2 nmol6-20 nmol12 h72 h24 h (24-48)6-7 dLiverFMT 680/700
IVIS 675/720
ProSense 750 FASTIV2 nmol6-20 nmol24 h72 h24 h6-7 dLow liver, intestineFMT 750/770
IVIS 745/800
ProSense 750 EXIV4 nmol12-40 nmol30 min36 h6-24 h
3 dLow liver, bladderFMT 750/770
IVIS 745/800

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Figure 2: A) Precise fluorescence localization of activated ProSense 680 is shown by FMT imaging. The non-invasive tomographic imaging is able to detect disease-related protease activity and accurate quantification of the internal distribution of this fluorescence in the lung. B) FMT measured a 5-fold increase in ProSense 680 fluorescence in the lungs of chronic obstructive pulmonary disease (COPD, asthma) mice (> 55 pmol/lung) as compared to those of control mice (< 10-15 pmol/lung). The FMT software precisely quantified the volume of the fluorescence within the lung regions of the mice to provide a measure of affected lung volume, showing approximately a 3-fold increase in COPD animals as compared to controls (~100 mm3 in asthma vs 30 mm3 in controls). Both the total fluorescence quantification (pmol) and the volume measurement (mm3) show high statistical differences in comparing asthma and control mice (p<0.0007 and p<0.002, respectively).

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Figure 3: Rolipram (a PDE4 inhibitor) has been proven as an anti-inflammatory drug. To evaluate whether the effect of a PDE4 inhibitor on COPD treatment could be detected and measured non-invasively by FMT, mice were treated with Rolipram (2 mg/kg given intraperitoneally 24, 12, and 0.5 hours prior to intranasal lipopolysaccharide treatment (LPS), which induces inflammation). Control mice received injections of PBS. All mice were injected with ProSense 680 4hours after LPS treatment. A) FMT showed high fluorescence signal in untreated asthmatic mice compared to Rolipram-treated mice. B) The quantification of the therapeutic response in treated LPS-induced lung inflammation showed 92% inhibition of total ProSense 680 signal and 86% inhibition of fluorescence volume.

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Figure 4: Tissue 2D imaging of ProSense 680 fluorescence was measured ex vivo to verify the dramatic effects of Rolipram treatment on LPS lung inflammation and on non-invasive FMT quantification. Image analysis to generate tissue fluorescence ratios to background yielded a highly significant (p < 0.001) inhibition of ProSense 680 tissue fluorescence by Rolipram treatment.

prosensefig5.jpg
Figure 5: BAL cell differential analysis indicates the expected recruitment eosinophils (> 60% of the total cells) infiltrating the airways of asthmatic mice and no detectable eosinophils in control mice. A) Fluorescent microscopy of the BAL cells isolated from asthmatic and control mice that received intravenous ProSense 680 injection. This data shows ProSense 680 fluorescence is predominantly activated by eosinophils cells in the airways. B) BAL mononuclear cells in the same mice showed little or no fluorescence, whereas BAL cells from control mice (predominantly macrophages) showed minimal fluorescence. C) Tissue sections from the lungs of asthmatic mice demonstrated multiple areas of fluorescence localized to the lung parenchyma. Minimal fluorescence is detectable in the control lungs.


Using ProSense in vitro


ProSense agents have been validated for use with tumor and inflammatory cells.

Flow cytometry and in vitro microscopy

We have validated ProSense agents for use with fluorescence microscopes and flow cytometers. Here is a brief protocol with a recommended concentration of agent to use:

  1. Culture cells in standard TC plate or chamber slide. 
  2. Incubate cells with 1 μM ProSense 680, 1-2 μM ProSense 750 EX, or 1 μM ProSense 750 FAST for 6-24 hours at 37 °C. 
  3. Wash 1x with PBS. For flow cytometry, detach and resuspend cells in PBS. 
  4. Flow cytometry filter settings: 712/21 (ProSense 680), 780/60 (ProSense 750 EX or ProSense 750 FAST).
    Fluorescence micrsocopy filter: Cy5.5 (ProSense 680), Cy5.5 or Cy7* (ProSense 750 EX or ProSense 750 FAST).

In Vitro Imaging

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Figure 6: Raw 264.7 mouse macrophages were incubated with ProSense 750 FAST (1 μM final concentration). The cell-permeable cysteine/calpain inhibitor was added 1 hour before addition of the agent. Cells were analyzed by fluorescence microscopy. ProSense 750 FAST is activated by RAW macrophages. The pan-cathepsin inhibitor E64d significantly reduces the activation.


Application notes and posters


  • Application Note: Quantitative Pre-clinical Fluorescence Imaging of Cancer Metastasis to the Lung and Response to Therapy 
  • Application Note: Noninvasive, In Vivo Quantitation of Asthma Severity using Fluorescence Molecular Tomography 
  • Application Note: Non-Invasive Quantitative In Vivo Imaging of Atherosclerosis Disease Progression and Treatment Response in ApoE Deficient Mice using Fluorescence Molecular Tomography and NIR Fluorescent Pre-clinical Imaging Agents


FAQs


Q. Can I use ProSense be used in humans?

A. No, ProSense is intended for animal research only and not for use in humans.


Q. What is the difference between ProSense 750 FAST and ProSense 750 EX?

A. ProSense 750 FAST is one of our F.A.S.T. (Fluorescent Activatable Sensor Technology) agents that confers an improved pharmacokinetic profile with earlier imaging time points. It is activated by Cathepsin B, L, S, K, V and D and is not cleaved by trypsin or plasmin. ProSense 750 EX is activated by Cathepsin B, L, S and Plasmin. ProSense 750 EX has a much faster clearance time (see above table).


Citations


Please visit our Citations Library for references using ProSense on the IVIS or on the FMT.