V24 - CSRP1 Suppression Promotes 3D Spheroid Invasion in Lung Cancer Models

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Ashley Urrutia Avila

PhD Candidate
University of California Irvine, United States

Co-Author(s)

Timothy L. Downing

Associate Professor
University of California Irvine
Irvine, California, United States

Primary Investigator(s)

Timothy L. Downing

Associate Professor
University of California Irvine
Irvine, California, United States

Last Author(s)

MD

Michelle A. Digman

Professor
University of California Irvine, United States

Introduction: : Lung cancer is the world's most lethal cancer, largely due to its highly metastatic nature. Studies demonstrate that reduced expression of focal adhesions, which comprise multi-protein structures that connect the cytoskeleton of a cell to their surrounding extracellular matrix (ECM), increases metastatic spread. Additionally, previous studies have found that disrupted cell attachment to the ECM promotes cell survival and invasion potential during metastasis. Preliminary findings from our group demonstrate that knockdown of the CSRP1 adhesome protein (CSRP1KD) enhances metastatic potential in somatic lung cancer cell lines in vivo. Notably, A549 cells required exposure to stem cell factors in the culture medium to exhibit this phenotype, whereas H1299 cells displayed increased metastatic behavior regardless of media conditions. Although significant, these preliminary studies require further investigation to better understand the cellular mechanisms driving this increase in metastatic potential upon CSRP1 KD. Thus, the aim of this study is to investigate the fundamental mechanism of metastasis within a simplified in vitro spheroid invasion model assay. We hypothesized that CSRP1KD, particularly in combination with stem cell media, will increase spheroid invasion.

Materials and

Methods: : To investigate this, somatic lung cancer cell lines (A549 and H1299) were treated via lentiviral transduction to knockdown the CSRP1 gene and were either fed with stem cell media or standard cell growth medium. To create spheroids, transduced cells were cultured in 96 well plates with ultra-low adhesion and spun down at 1000 rpm for 10 minutes. After 3 days, cells were then transferred into matrigel to mimic the tumor microenvironment. Once transferred cells were imaged over the course of 6 days. These images were then outlined using a custom FIJI ImageJ code to quantify spheroid invasion area.

Results, Conclusions, and Discussions:: It was determined that CSRP1KD within both lung cancer cell lines (A549 and H1299) enhanced spheroid invasion. A549 cells required exposure to stem cell factors in the culture medium to exhibit this effect, whereas H1299 cells displayed heightened metastatic potential regardless of extrinsic media conditions. These findings are in line with our in vivo experiments and align with broader evidence implicating impaired focal adhesion dynamics in facilitating metastasis. To build on these findings, future work will evaluate whether CSRP1KD activates epithelial-to-mesenchymal transition (EMT) programs in spheroids by analyzing molecular markers (e.g., E‑cadherin, N‑cadherin, vimentin). Additionally, we will explore the involvement of mechanotransduction pathways such as RhoA/ROCK or FAK signaling in driving invasion.

Acknowledgements and/or References (Optional): :