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Optimizing Tissue Collection for PD Assays Using Non-Clinical Models

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

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Optimizing Tissue Collection for PD Assays Using Non-Clinical Models

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

  2. Typical Rodent Sample Collection Methods • Euthanasia (CO2) • Tumor resection • Transfer to tube • Freeze in dry ice or -70C freezer • Timing has not traditionally been perceived as crucial

  3. 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

  4. Comparing All Four Harvest Methods on the Same Gel +C C011 Untreated Jurkats Resection After CO2 SAC Resection After CO2 SAC Cryobiopsy + Anesthesia Cryobiopsy + Anesthesia Resection + Anesthesia Resection + Anesthesia pAKT Settings Min 20.0 Max 75.0 1 min exp. FNA + Anesthesia 200 FNA + Anesthesia 140 100 80 Actin Settings Min 20.0 Max 3000.0 30 sec exp. 60 kDa 60 50 40 30 b-Actin phospho-AKT (Cell Signaling #9271) Separated on an 8% Tris-Gly Gel

  5. 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.

  6. 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).

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

  8. Pre-chill cryovials in liquid nitrogen

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

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

  11. Prechilled tube is withdrawn from liquid nitrogen

  12. 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.

  13. Examples of xenograft tumor biopsies

  14. Inconsistency of Excisional Biopsies • The first two images show frozen sections of excisional biopsies (H&E). • The third image shows a needle biopsy (H&E).

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

  16. 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

  17. The Next Speaker is:Dr. Robert Kinders

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