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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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Prechilled tube is withdrawn from liquid nitrogen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

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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