Image-guided extraction of invasive cancer cells via photomarking Open Access

Zohbi, Najdat (Spring 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/br86b3588?locale=en
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Abstract

Intratumoral heterogeneity is a major obstacle to understanding the biological drivers of tumor progression and greatly undermines the effectiveness of cancer therapeutics causing relapse and poor clinical outcome. In particular, phenotypic heterogeneity can be observed in cancer cell populations. To assess how such heterogeneity drives tumor progression, our lab developed an imaging-based technique referred to as Spatiotemporal Genomic Analysis (SaGA) allowing us to isolate, extract, and maintain rare cells exhibiting distinct phenotypes for genetic profiling. Previously, SaGA has been used to cultivate purified leader and follower cell lines from collectively invading 3D models. Isolation of these distinct subpopulations is achieved through two-color based flow cytometry, which in turn relies on differential fluorescence of photoactivatable fluorophores. We propose two methods in which this powerful technique can be optimized and expanded to other cell lines that collectively invade. One approach to streamlining the SaGA process entails the use of a cell-permeable, non-cytotoxic photoconvertible fluorescent dye referred to as (E)-3/(Z)-3. Optimization of this dye was done within H1299 lung adenocarcinoma models to recapitulate our previously established leader and follower lines generated by the photoswitchable protein Dendra2. An alternative method takes advantage of the nuclear localization of Dendra2 which involves tagging histone2B (H2B) in target cells. The use of this H2B-Dendra2 alternative was explored in 4T1/4T07 murine mammary adenocarcinoma models for their phenotypically distinct mode of collective invasion. Here, we detail the technical and biological aspects of optimizing and expanding our SaGA technique using these systems.  

Table of Contents

Table of Contents

INTRODUCTION.. 1

Overview of Metastatic Heterogeneity. 2

Collective Invasion and Cooperation in Cancer 4

Spatiotemporal Genomic and Cellular Analysis (SaGA) 6

Expanding SaGA with the H2B-Denra2 Photoactivatable Protein. 12

Scope of the Thesis. 15

MATERIALS AND METHODS. 16

Cell lines and transductions. 17

Generation of 3D tumor spheroids. 18

Staining with E/Z-3. 18

MTT Assay. 19

Image and spheroid analysis. 20

Seeding and embedding spheroids. 20

Photoconversion. 20

Matrix degradation. 21

Fluorescence activated cell sorting (FACS) 22

RESULTS. 23

H1299 cells are stainable with E/Z-3 with minimal cytotoxicity. 24

Photoconversion of E/Z-3 produces a significantly detectable signal 26

H1299 leaders and followers are discernible and convertible in stained spheroids. 29

Photoconverted H1299 leaders and follower subpopulations are isolated via FACS. 30

Purified H1299 leaders and followers maintain their respective phenotypes post-sort 32

H2B-dendra2 4T1/4T07 cells are photoconvertible with high specificity. 33

Photoconverted H2B-dendra2 4T1/4T07 cells are isolated via FACS. 35

4T1 leader cells are highly invasive and exhibit increased single cell invasion. 38

DISCUSSION.. 41

Future Directions. 47

REFERENCES. 49

APPENDIX.. 51

 

 

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