R26 - CSRP1 Knockdown and Stem Cell Media Promote Spheroid Compaction, Suggesting Enhanced Metastatic Potential in Various Lung Cancer Cell Lines

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Daisy Cortez

Undergraduate
University of California, Irvine
Santa Ana, California, United States

Primary Investigator(s)

MD

Michelle A. Digman

Professor
University of California Irvine, United States

Co-Author(s)

Ashley Urrutia Avila

PhD Candidate
University of California Irvine, United States
Timothy L. Downing

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

Last Author(s)

Timothy L. Downing

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

Introduction: : Lung cancer has the highest cancer-related mortality rate worldwide, largely due to its high metastatic potential. The adhesome, a dynamic network of structural proteins and signaling pathways that regulate cell adhesion and cytoskeletal interactions, plays a critical role in metastasis. Prior studies have shown that reduced adhesome expression promotes tumor progression. Preliminary findings from our lab demonstrate that knockdown of the CSRP1 adhesome protein (CSRP1KD) enhances metastatic potential in somatic lung cancer cell lines. 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.

To further investigate this relationship, we employed an in vitro tumor spheroid model to study spheroid compaction, a process associated with cell cohesion, invasiveness, and metastatic potential. We hypothesized that CSRP1KD, particularly in combination with stem cell media, would increase spheroid compaction.

Materials and

Methods: : Lung cancer cell lines A549, H460, H1299, and Calu-3, differing in p53 expression status and site of origin, were used. A549, H460, and Calu-3 cells express wild-type p53, while H1299 cells are p53-null. A549 and H460 originate from primary lung tumors, whereas H1299 and Calu-3 are derived from metastatic sites. Spheroids were formed in ultra-low adhesion 96-well plates, centrifuged, and cultured for three days. Daily brightfield images were collected and analyzed using a custom FIJI (ImageJ) pipeline to quantify spheroid area and circularity.

Results, Conclusions, and Discussions:: Our results showed that CSRP1KD combined with stem cell media significantly enhanced spheroid compaction in A549, H460, and Calu-3 cell lines, as indicated by reduced spheroid area and increased circularity relative to scramble controls. Interestingly, in the H1299 cell line, which is p53-null and derived from a metastatic site, CSRP1KD alone led to the greatest spheroid compaction, independent of media conditions. These findings suggest that CSRP1 disruption can potentiate features associated with metastasis, and that this effect may be modulated by the intrinsic properties of each cell line, such as p53 status and tumor origin.

Overall, our data support a role for adhesome disruption and stem cell-like conditions in promoting spheroid compaction, reinforcing the association between compaction and metastatic potential. Future studies will explore the role of additional adhesome genes in this process to further elucidate the link between focal adhesion dynamics and lung cancer progression.

Acknowledgements and/or References (Optional): :