Optimizing Tissue Collection for PD Assays Using Non-Clinical Models

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Optimizing Tissue Collection for PD Assays Using
Non-Clinical Models

• Melinda Hollingshead, DVM, PhD

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(No Transcript)
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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

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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

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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.
200
FNA Anesthesia
FNA Anesthesia
140
100
80
Actin Settings Min 20.0 Max 3000.0 30 sec exp.
60 kDa
60
50
40
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b-Actin
phospho-AKT (Cell Signaling 9271) Separated on
an 8 Tris-Gly Gel
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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.
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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).
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So, how are we collecting PD samples from
preclinical efficacy models?
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Pre-chill cryovials in liquid nitrogen
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Mouse is anesthetized with isoflurane inhalation
anesthetic, tumor is wiped with disinfectant
The biopsy needle is cocked to prepare the sample
collection chamber
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The biopsy is collected percutaneously or by open
exposure depending upon the experimental goal
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Prechilled tube is withdrawn from liquid nitrogen
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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.
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Examples of xenograft tumor biopsies
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Inconsistency of Excisional Biopsies

• The first two images show frozen sections of
  excisional biopsies (HE).
• The third image shows a needle biopsy (HE).

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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 11
Mouse 13
Mouse 18
Mouse 16
Mouse 17
Mouse 19
Mouse 21
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 SC Tumors Collected Post Dose 4
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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

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The Next Speaker isDr. Robert Kinders