Cryoinjury of Breast Cancer Cells in Tissue Equivalent Constructs

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1 Cryoinjury of Breast Cancer Cells in Tissue Equivalent Constructs Bumsoo Han Department of Mechanical and Aerospace Engineering University of Texas at Arlington

2 Cryosurgery Minimally invasive surgical destruction of malignant or unwanted tissues by localized freezing Freezing by a fine surgical probe cooled with compressed argon (Joule-Thomson Effect) Intraoperative imaging provides surgeons with the progress of surgery (US, MRI and CT) Breast Cancer (Sanarus Medical) Prostate Cancer (Endocare & Oncura)

3 Challenges in Cryosurgery Incomplete killing zone between iceball edge and effective destruction zone Requirement of surgical margin to ensure complete tumor destruction Limited ability of monitoring effective destruction zone Establish thermal conditions of complete tissue destruction for various diseases. Make incomplete killing zone smaller by enhancing freezing injury within it.

4 Cryosurgical Injury Models Models Method Advantages Disadvantages Cell suspension Suspending cells in proper media Highest controllability No C-C nor C-ECM interaction Cell monolayer Monolayer or clones of cells on a substrate High controllability C-C interaction Cell-substrate interaction No C-ECM interaction No 3D effect Engineered artificial tissue (Present study) Seeding cells within a collagen gel matrix High controllability C-C interaction C-ECM interaction Partial 3D effect No vascular structure In vitro native tissue Harvesting tissue from a host animal C-C interaction C-ECM interaction 3D effect Medium controllability Non-functional vascular structure 2D in vivo tissue Growing tumor at the skin of a host animal (i.e. dorsal skin fold chamber) C-C interaction C-ECM interaction 3D effect Partial vascular structure Medium controllability No large blood vessels 3D in vivo tissue Growing tumor within a host animal C-C interaction C-ECM interaction 3D effect Full vascular structure Low controllability

5 Engineered Tumor Tissue MCF7 human breast cancer cells are seeded in type I collagen matrix and cultured. The cells grow within the collagen matrix. The cells are attached to collagen fibers which are working as extracellular matrix. Cell-cell, cell-ecm interaction, and cellbased healing response

6 Cryosurgery Simulator Thermocouple Cryosurgery Probe Upper Jig Jig Supporter Lower Jig Petri Dish Culture Media Engineered Tumor Tissue

7 Thermal History and Prediction during A Simulated Surgery Temperature ( C) Measurement Numerical model r=6mm r=6mm r=11mm r=11mm r=16mm r=16mm r=21mm r=21mm Time (sec) Numerical analysis based on one dimensional conduction equation solved by an enthalpy method Generally speaking, measured temperature profiles agree well with numerical prediction. Estimated edge of the ice ball 16mm

8 Freezing Injury of MCF7 in TEs Viability (%) Temperature ( C) Day 0 Day 1 Day Edge of Iceball Radial Location (mm) Radial Location (mm) Day 0 (3 hours) Less than 20% survival of MCF7 within 11mm No or minimal effects at the unfrozen locations Day 1 & 3 Tumor cells repopulate at the frozen locations. At 11mm, the tumor cell viability returns to the level of the unfrozen state. Tumor cell repopulation can be caused by cell reproduction and migration. Upper Fig - Post-thaw viability Lower Fig - End temperature

9 Histology - Complete Killing Zone Location: 3mm from the center of the probe Tapered probe site Mainly non-viable cells with hyperchromic nuclei Viability < 5% 10x Hypo-cellular density Rare voids in the matrix 40x

10 Histology - Incomplete Killing Zone Location: 11mm from the center of the probe Mixed viable and damaged cells Viability: 30 ~ 60% Moderate cellular density Numerous voids in the matrix It has a stranded appearance with increased staining suggesting concentrated collagen. 10x 40x

11 Histology - Live Zone Location: 18mm from the center of the probe Intact matrix structure Mainly viable cells Viability > 95% Highest cellular density 10x 40x

12 Tumor Necrosis Factor - α (TNF- α) Proinflammation cytokine Upregulate apoptotic signal pathways of tumor cells (caspases activation) At certain conditions, upregulate proliferation pathways of tumor cells (NF-kB activation) Toxic at high dosage Need to find a way to maximize the effectiveness with minimizing the toxicity

13 Effects of TNF-α treatment only Treatment with TNF-α Concentration : 100ng/ml Duration: 24 hours Cell number TNF-a treatment may stimulate the proliferation but the effect is not statistically significant. Viability No notable viability drop by the concentration and duration of current TNF-α treatment Cells/field Viability (%) TNF-! Treatment Control Day 0 Day 1 Day 3 Time Point TNF-! Treatment Control Day 0 Day 1 Day 3 Time Point

14 Freezing Injury with TNF-α Viability (%) Viability (%) Day 0 Day 1 Day Control Day 0 Day 1 Day 3 Radial Location (mm) Radial Location (mm) Day 0 Less than 10% survival of MCF7 within 11mm TNF-a slightly stimulates cell growth at the unfrozen locations. Day 1 & 3 Tumor cell repopulation is significantly inhibited at all frozen locations. At 11mm, the viability remains around 40% Upper Fig - Viability w TNF-α Lower Fig - Viability w/o TNF-α

15 Summary and Conclusion A cryoinjury model by use of tissue engineering technology was developed and its capability was demonstrated. Cryoinjury characteristics of human breast cancer cells were investigated. TNF-α was very effective to inhibit repopulation of MCF7 cells after cryosurgery. Future Research Application of current model to other cell lines Structural change due to freezing

16 Acknowledgements Matthew D. Egberg, Pung Haung David Swanlund James E. Coad, MD John C. Bischof, PhD Funding US DOD BCRP DAMD to BH BMEI Thermal Therapy Interest Group to JB

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