Cancer metastasis, the spread of cancer cells from a primary tumor to distant organs, is the leading cause of cancer-related deaths. During metastasis, cancer cells undergo significant changes in their mechanical properties, including alterations in cell elasticity. Cancer cells generally exhibit higher elasticity than normal cells, a key feature that may contribute to their ability to migrate and invade other tissues. Although extensive research has focused on cell-substrate interactions, these studies do not fully replicate the physiological environment, where cells are frequently in direct contact with each other. In this study, Atomic Force Microscopy (AFM) in force spectroscopy mode, utilizing a micro-sized cantilever with a hemispherical tip, was used to quantify the Young’s modulus (E) of MCF-7 breast cancer cells in two configurations: cell-substrate and cell-cell. The resulting force-indentation curves were analyzed and fitted to the Hertz contact model to determine the Young’s modulus. The results showed that the Young’s modulus in the cell-cell configuration was Ecc = 205.8 ± 50.88 Pa, while in the cell-substrate configuration, it was Ecs = 187.95 ± 78.26 Pa. These findings suggest that MCF-7 cells are slightly less elastic in the cell-cell configuration. This challenges the expectation of a more significant difference between the two configurations and highlights the importance of considering biological variability and experimental conditions when interpreting cell mechanical properties.