Defining the clinical and biological relevance of leader and follower cell mutations in collective cancer invasion Open Access

Pedro, Brian (Spring 2020)

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Cancer metastasis, the direct cause of 90% of cancer-related deaths, remains a poorly-understood process. Metastatic carcinomas often utilize collective invasion, whereby cohesive packs of cells travel through the microenvironment. Furthermore, in vitro studies of lung cancer collective invasion have revealed that specialized leader and follower cells cooperate to facilitate the emergence of invasive chains, and that follower cells cannot invade in the absence of leaders. However, the biology underlying these phenotypes, including the role of gene mutations, has not been fully explored. We discovered novel leader-specific and follower-specific gene mutations, including, notably, a leader-specific mutation in ARP3. Introduction of this mutation into follower cells conferred leader-like behavior, including the ability to lead invasive chains, suggesting it could play an important role in driving leader cell emergence and behavior.

There is currently a lack of predictive biomarkers for determining high-risk patients in a number of cancer types, including lung cancer. Even among patients diagnosed with localized disease, over 40% are not expected to survive beyond five years. We thus investigated whether high-risk patients could be identified by the presence of mutations within a leader-cell derived cluster of genes on chromosome 16q. Using cohorts of lung squamous cell carcinoma and lung adenocarcinoma patients from The Cancer Genome Atlas, we found poorer survival for 16qMC+ patients; furthermore, this correlation was observed among two cohorts of hepatocellular carcinoma patients, another cancer type with high rates of metastasis and disease recurrence.

Finally, we combined SaGA with single-cell RNA-sequencing to further dissect the biology of leader and follower cells during active collective invasion. We discovered that leader and follower mutational profiles are mutually exclusive on the single cell level. These mutations also correlate strongly with leader and follower expression markers including MYO10 and IL13RA2, indicating that these mutations could be utilized as precise genomic markers for individual leaders and followers. We further discovered that leader cells harbor cancer stem cell-like gene expression, and that TGFβ signaling may facilitate leader-follower cooperation. Ultimately, these data demonstrate that leader- and follower-specific mutations can both elucidate the mechanisms of collective invasion and help to better stratifying high-risk cancer patients.

Table of Contents

Chapter 1: Introduction 1

1.1. Overview of cancer metastasis 1

1.1.1. Statistics and clinical implications of cancer metastasis 1

1.1.2. Steps of the metastatic cascade 4

1.1.3. Single and collective cell migration and invasion 4

1.2. Overview of tumor heterogeneity 8

1.2.1. Clonal evolution of tumor subpopulations 8

1.2.2. Phenotypic heterogeneity 9

1.2.3. Leader-follower dynamics in cancer cell invasion 13

1.3. Dissertation goals 15

Chapter 2: Genetic heterogeneity within collective invasion packs drives leader and follower cell phenotypes 17

Abstract 19

2.1 Introduction 20

2.2 Materials and Methods 23

2.3 Results 29

2.4 Discussion 46

Supplementary Information 50

Chapter 3: Prognostic significance of an invasive leader cell-derived mutation cluster on chromosome 16q 53

Abstract 54

3.1 Introduction 55

3.2 Methods 57

3.3 Results 60

3.4 Discussion 73

Supplementary Information 76

Chapter 4: Single-cell RNA-sequencing of lung cancer leader and follower cells reveals distinct mutational profiles and cancer stem cell-like gene expression patterns 87

4.1 Introduction 88

4.2 Materials and Methods 90

4.3 Results 94

4.4 Discussion 111

Supplementary information 116

Chapter 5: Conclusions and Future Directions 118

5.1 Role of gene mutations in dissecting leader and follower cell biology 119

5.2 Clinical implications of the leader-derived 16q mutation cluster 121

5.3 Characterization of leader cells as a cancer stem cell-like population and the role of TGFβ signaling in leader-follower cooperativity 123

References 127

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