Optimizing Tissue Collection for PD Assays Using Non-Clinical Models Melinda Hollingshead, DVM, PhD
Optimizing Tissue Collection for PD Assays Using Non-Clinical
Models
Melinda Hollingshead, DVM, PhD
Typical Rodent Sample Collection Methods
• Euthanasia (CO2)
• Tumor resection
• Transfer to tube
• Freeze in dry ice or -70C freezer
• Timing has not traditionally been perceived as crucial
Effect of Sampling Procedures on Protein Levels in Tumors
• Tumors in mice were sampled as follows:– Cryobiopsy + Anesthesia– Resection + Anesthesia– Fine Needle Aspirate (FNA) + Anesthesia
– Resection after CO2 Sacrifice
– Measured ERK, AKT, MEK1/2 & Phospho Proteins
pAKT SettingsMin 20.0Max 75.01 min exp.
phospho-AKT(Cell Signaling #9271)
Separated on an 8% Tris-Gly Gel
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b-Actin
Actin SettingsMin 20.0
Max 3000.0 30 sec exp.
Comparing All Four Harvest Methods on the Same Gel
The cryobiopsy device leaves a 10-gauge wound in the
sampled tissue. We sought to reduce the biopsy diameter so we could do repeat samples in
mice and transfer the technique to the clinic.
Dorsal and lateral views of the biopsy needle sample chamber. This chamber is opened by cocking the needle prior to inserting it into the tissue. These biopsy needles are available in a variety of sizes (14-22 gauge).
So, how are we collecting PD samples from
preclinical efficacy models?
Pre-chill cryovials in liquid nitrogen
The biopsy needle is cocked to
prepare the sample
collection chamber
Mouse is anesthetized
with isoflurane inhalation anesthetic,
tumor is wiped with disinfectant
The biopsy is collected
percutaneously or by open exposure
depending upon the
experimental goal
Prechilled tube is withdrawn from liquid nitrogen
Sample is transferred from needle to prechilled cryovial by touching the tumor sample to the inner wall. The
biopsy adheres to the tube and rapidly freezes.
Examples of xenograft tumor biopsies
• The first two images show frozen sections of excisional biopsies (H&E).
• The third image shows a needle biopsy (H&E).
Inconsistency of Excisional Biopsies
Colo-829 SC Tumors Collected Post Dose 4
Poly-ADP-ribose (Trevigen)Min 20.0
Max 400.04 min exp.
Drug Vehicle Q12h x 5D PO
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ABT-888 25 mg/kg Q12h x 5D PO
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Use of Frozen Tumor Samples for Assessing PD
Summary
• Choice of collection methods can profoundly affect target analysis outcomes
• Mouse xenografts help optimize sample collection and handling protocols
• Comparisons of experimental variables should aid in determining optimum sample handling
• Xenografts provide samples that simulate clinical material during assay development
• Preclinical models can be designed to use/test clinically relevant methods
The Next Speaker is:
Dr. Robert Kinders