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Cancer Technologies

Cancer Technologies - Poster Session E

Poster S13 - Clustering Enchances Cancer Cell Survival in Extreme Extracellular Conditions

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Antitumor drug resistance and tumor relapse are the primary factors that lead to mortality in cancer patients [1]. Tumors developing resistance enter a state of metabolic dormancy in vivo that can remain undetectable clinically and allow for resumed growth later in the form of remission [2, 3]. Larger tumors have seen worse prognosis clinically in cancer patients compared to smaller tumors. Circulating tumor clusters, a byproduct of metastasis, undergo adaptive changes to their metabolism, enhancing their ability to survive in harsh circulatory microenvironments versus individual circulating tumor cells [4]. Previous studies have identified that extreme conditions like shear stress in circulation, hypoxia, low nutrition, drugs, and radiation can contribute to metastasis or dormancy in cancer [5, 6, 7]. While recent studies have elucidated some of the factors related to the maintenance and escape from growth arrest [8], few studies critically analyze physical factors in the tumor microenvironment that lead to cell dormancy or otherwise focus on cell survival in response to extracellular factors, or even how adaptive metabolism may contribute to these behaviors. Our group sought to determine how clustering may enhance cancer cell survival upon extreme changes to the microenvironment, which may lead to cancer cells entering dormancy.

Materials and

Methods:  4T1 murine breast cancer cells were seeded onto 8% linear polyacrylamide-patterned micropatterned coverslips (Fig 1A, 1C), adapted from the protocol as described in [9], containing circular patterns ranging from 225 microns (~500 cells per cluster) to 11.3 microns (~1 cell per cluster) radii to control cluster size. Once cells reached confluency in the patterns, cells were either cultured in a control culture media of RPMI1640 containing 10% FBS and 25 mM Glucose or were treated with a starvation media of either 0% FBS or 0 mM Glucose. After 24 hours of treatment, cells were stained with a FITC annexin-V apoptosis assay and a Hoechst 33342 nuclear counterstain (Fig 1B). Cells were then fixed with a 4% paraformaldehyde solution and imaged using a Zeiss LSM 900 Confocal Microscope. To calculate cellular response to the treatments, an “apoptotic ratio” was calculated by dividing the number of early apoptotic cells (as stained by Annexin-V) by the total number of cells in a cluster (as stained by the counterstain).

Results, Conclusions, and Discussions: As the cluster size increased, apoptotic rates for cells in both serum starvation and glucose deprivation decreased, whereas cluster size had a minimal impact on the survival of cells in control conditions (Fig 1D, 1E). When comparing both treatments to the corresponding control conditions, large clusters (225 μm radii) exhibited almost identical low apoptotic rates, whereas smaller clusters in extreme conditions experienced a significant increase in apoptotic ratio compared to their control state (Fig 1F, Fig 1G). Both instances together indicate that some characteristics of larger clusters contribute to increased resilience to extreme extracellular conditions that may improve long-term resistance to cancer therapy. It has been documented in past clinical research that larger tumors appear to have a poorer prognosis and may have an increased chance of remission after chemotherapy [4]. Additionally, some data in our recorded set indicates higher cell numbers improve survival on average within sizes of the smaller clusters (28.2 – 16.9 μm radii), which may point to cell number being one key factor for cluster survival. Past research also indicates that differences in metabolic states of a tumor may contribute to start signals for metastasis or otherwise kickstart growth in dormancy [7, 8]; it has also been documented that basal metabolic patterns tend to vary between migrating/non-migrating tumors [10]. Given these past results, our group is currently working to analyze how glucose uptake varies between these cluster sizes in starvation conditions using 2-NBDG and to examine how mitochondrial membrane potential may vary using TMRM. We also intend to utilize 3D spheroids to increase physiological relevance.

Clustering plays a beneficial role in cancer survival during starvation. Additionally, the number of cells within clusters may be a key factor in cell survival and potential for remission. Past research indicates that differences in metabolism may be contributing factors to survival. Future work entails robust testing of glucose uptake, mitochondrial membrane potential, and redox ratio analysis to determine how metabolism may contribute to cell survival. Additionally, we intend to translate our findings into 3-dimensional spheroids in collagen matrices to establish a model that is closer to in vivo behavior.

Acknowledgements (Optional):  This work was supported by the National Institute of Health Grants (GM147958 and EB034930) and a pilot fund from Arkansas from the Arkansas Integrative Metabolic Research Center (to J.Z.).

References:
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