1. Optimizing Tissue Collection for PD Assays Using Non-Clinical Models Melinda Hollingshead, DVM, PhD

3. Typical Rodent Sample Collection Methods Euthanasia (CO 2 ) Tumor resection Transfer to tube Freeze in dry ice or -70 C freezer Timing has not traditionally been perceived as crucial

4. 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 CO 2 Sacrifice –Measured ERK, AKT, MEK1/2 & Phospho Proteins

5. pAKT Settings Min 20.0 Max 75.0 1 min exp. phospho-AKT (Cell Signaling #9271) Separated on an 8% Tris-Gly Gel 200 140 100 80 60 50 40 30 FNA + Anesthesia Cryobiopsy + Anesthesia Resection + AnesthesiaResection After CO 2 SAC Cryobiopsy + AnesthesiaResection + Anesthesia FNA + AnesthesiaResection After CO 2 SAC +C C011 Untreated Jurkats 60 kDa b-Actin Actin Settings Min 20.0 Max 3000.0 30 sec exp. Comparing All Four Harvest Methods on the Same Gel

6. 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.

7. 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).

8. So, how are we collecting PD samples from preclinical efficacy models?

9. Pre-chill cryovials in liquid nitrogen

10. The biopsy needle is cocked to prepare the sample collection chamber Mouse is anesthetized with isoflurane inhalation anesthetic, tumor is wiped with disinfectant

11. The biopsy is collected percutaneously or by open exposure depending upon the experimental goal

12. Prechilled tube is withdrawn from liquid nitrogen

13. 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.

14. Examples of xenograft tumor biopsies

15. 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

16. Colo-829 SC Tumors Collected Post Dose 4 Poly-ADP-ribose (Trevigen) Min 20.0 Max 400.0 4 min exp. Drug Vehicle Q12h x 5D PO Mouse 5Mouse 9Mouse 11Mouse 13 Mouse 15 Mouse 18 AAXR2-7 #1 200 140 100 80 513 b-Actin ABT-888 25 mg/kg Q12h x 5D PO Mouse 3Mouse 7Mouse 16Mouse 17Mouse 19Mouse 21 AAXR2-7 #1 517 Use of Frozen Tumor Samples for Assessing PD

17. 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

18. The Next Speaker is: Dr. Robert Kinders