Characterization of Jagged1-Notch signaling in collective invasion and determination of ALCAM expression in non-small cell lung cancer models Open Access

Harrison, Zachary (Spring 2019)

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Non-small cell lung cancer (NSCLC) accounts for 85% of new lung cancer diagnoses and patients diagnosed with the disease typically have poor prognoses. Intratumoral heterogeneity complicates NSCLC treatment, which adds to the poor outcomes for patients. Designation of subpopulations of more-invasive leader cells and growth-promoting follower cells in these heterogenous populations is possible. This study sought to identify and characterize Jag1 as a biomarker for NSCLC leader cells and elucidate the function of Jag1 in collective invasion. In addition, ALCAM expression was determined in leaders, followers, and heterogenous patient-derived primary cells to assess biomarker potential. Cell cycle analysis by flow cytometry was performed on H1299 Jag1+ and Jag1-knockdown cells to determine if Jag1 expression is correlated with cell cycle progression. No difference was observed in cell distribution in the cell cycle stages in relation to Jag1 expression status. Next, a simple proliferation assay was performed on H1299 Jag1+ and Jag1-knockdown cells to investigate if Jag1 expression alters growth kinetics. Again, no difference was observed in the rate of proliferation between Jag1-expressing and Jag1-knockdown H1299 cells. ALCAM expression was determined in H1299 leaders and followers, in addition to heterogenous patient-derived primary cells, to determine if it is a biomarker for more invasive NSCLC subpopulations. ALCAM expression was high in H1299 leader cells, while it was low in followers. Interestingly, both patient-derived primary cell types analyzed, EUH 3123 and EUH 3174, exhibited high ALCAM expression, despite variable invasive potential in vitro. Despite no observed difference in cell cycle distribution or growth kinetics, it appears that Jag1 is a biomarker for H1299 leader cells on the basis of collective 

invasion data not shown here. ALCAM, with high expression in two primary cell types that exhibit different invasion patterns, does not appear to be a biomarker for more invasive NSCLC subpopulations. Further studies are needed to fully characterize the role of ALCAM signaling in NSCLC collective invasion. 

Table of Contents

I. Introduction 1

a. Lung cancer

b. Intratumoral heterogeneity: leaders and followers

c. Jag1-Notch signaling

d. Sustained proliferative signaling

e. ALCAM signaling

f. Specific aims and hypothesis

II. Cell cycle analysis of Jag-1-expressing and Jag-1 knockdown cells 6

a. Specific aim I

b. Rationale

c. Methods

i. Cell line

ii. shRNA knockdown

iii. Cell cycle analysis by flow cytometry

d. Results

III. Proliferation of Jag-1-expressing and Jag-1-knockdown cells 9

a. Specific aim II

b. Rationale

c. Method

i. Proliferation assay

d. Results

IV. Analysis of ALCAM expression in a non-small cell lung cancer cell lines and primary cell lines 11

a. Specific aim III

b. Rationale

c. Methods

i. Cell line and antibodies

ii. Flow cytometry

d. Results

V. Discussion and Conclusions 14

VI. References 19

VII. Figures 22

a. Figure 1: Cell cycle analysis of H1299 Jag1+ and Jag1-knockdown cells 

b. Figure 2: Proliferation analysis of H1299 Jag1+ and Jag1 knockdown cells 

c. Figure 3: ALCAM expression in H1299 subpopulations, EUH 3123, and EUH 3174 

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