20 Febraury 2019 |       Share
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Myelotoxicity in vivo by imaging

an innovative, rapid assay at the cost of an in vitro test

The measurement of the myelotoxicity is a routine test for anti-cancer NCEs. The conventional procedure includes a number of steps of growing complexity:

- in vitro analysis of the clonogenic potential of hematopoietic progenitors cells
- in vivo acute and chronic studies
- histological analysis of bone marrow cells isolated from control and treated mice  

A complete analysis is costly, requires time, qualified technical skills and the use of a large number of animals.

By exploiting the power of in vivo imaging, TOP srl offers a single step, sensitive and reproducible myelotoxicity assay at the same cost of an in vitro assay.

The assay developed by TOP srl is based on the  use of a transgenic mouse, the repTOPTMmitoIRE, engineered to express in all cells  the luciferase reporter under the control of a promoter activated in mitotic cells (Goeman et al, Molecular Biology of the Cell, 2012). In this mouse, it has been demonstrated that photon emission is directly proportional to cell proliferation, thus the effect of any anti-cancer drug can be measured by whole body imaging of living mice providing a direct, temporal measurement of the drug effects.

The major advantages of TOP srl assay versus conventional procedure are:

- a significant reduction of research time
- a significant cutback in costs
- the direct, in vivo quantification of the dose effect
- a detailed temporal analysis of the extent of myelotoxicity
- an accurate measurement of the time necessary for bone marrow recovery
- the full compliance of the 3R regulations with a significant reduction 
of the number of animals to be used and their distress.

 In vivo imaging for the rapid, direct, measurement of myelotoxicity.

In the repTOPTMmitoIRE mice the proliferation of bone marrow cells can be visualized and measured in vivo by simply taking a picture of the anaesthetized mouse with a CCD camera.  The extent of the effect of the treatment is directly proportional to the photon emission measured in the sternum or femur.

With this assay, for the first time, it is possible to monitor the effect of a given compound in time in the same animal and to define the time necessary to block cell proliferation and the physiological recovery  from the toxic treatment at any drug dosage.

a. The CCD camera pseudoimage shows the intensity of photon emission of repTOP™mitoIRE mice prior and after treatment with a single i.v. injection of a myelotoxic compound. b. The measurement of photon emission in the sternum and femur shows that after 2 days of treatment cell proliferation is strongly inhibited (** p<0.01). c. Daily imaging shows the recovery of bone marrow proliferation after a single dose treatment.

repTOP™mitoIRE mice were treated with 5-fluorouracil (5-FU) (150 mg/kg) or its vehicle. 5 days after treatment luciferase activity was measured by in vivo imaging (a.) and luciferase enzymatic activity in protein extracts from bone marrow (b.); bone marrow proliferating cells were measured by FACS (c.) The inhibition of luciferase activity measured in the repTOPTMmitoIREmouse model was directly correlated with the bone marrow cells in the S phase of the cell cycle.

Short term, long term effects: all-in-one.

The direct measurement of photon emission in living mice allows the quantification of the effects of the drug in study in a single animal in time: this facilitates the measurement of the extent of the effect of a given dose in terms of depletion of proliferating bone cells as well as the recovery of proliferation in time. Here below, the docetaxel myelosuppressive effects measured by in vivo imaging confirm the conventional toxicological studies described in the scientific literature (Bissery MC, European Journal of Cancer, 1995; Andrè S et al., Cell Pharmacol 1993) and in the EMEA dossier  (Procedure No.EMEA/H/C/1107 ).

repTOP™mitoIRE mice were treated with docetaxel (single i.v.) at three different doses. The daily measure of photon emission from sternum provides a quantitative evaluation of the inhibitory effects on bone marrow cells proliferation of docetaxel: at day 3 it was quite evident the dose dependent decrease of proliferation. The immunosuppression was recovered at different time points (depending on the dose).




Myelosuppressive effects of:

- new Chemical/Biological Entities

- new combinations of therapeutics

- novel formulation and drug delivery systems aimed at reducing toxicity of anticancer drugs



Types of assay available:

- Short term evaluation
- Long term evaluation  (recovery)
- Single and multiple treatment
- Drug Combination Study