In cancer pathologies, intratumor heterogeneity can be described as the co-localization of multiple malignant cell populations, sharing the same genotype but differing in their phenotypes. Since intratumoral heterogeneity is playing an extremely relevant role in chemotherapy, the study of this malignant hallmark has called the attention of the scientific community.

In this work, we hypothesize that cell heterogeneity is mostly triggered by mechanical stress, likely due to cell-cell fusion, incomplete cell segregation, or asymmetric division. To test our hypothesis, MDA-MB-231 breast cancer cells were cultured on 2D flat surfaces (i.e. cell culture flasks), being compared in terms of morpho-mechanical heterogeneity to cells cultured in polymer-based artificial tumors having an approximate elasticity of 25, 70 and 100 kPa (measured as Young’s Moduli). Artificial tumors correspond to alginate-gelatin microcapsules (600 µm in diameter), mimicking the mechanical properties of in vivo tumors (up to 100 kPa, measured as Young Moduli). After 7 days post immobilization, cells were isolated from their confined environment, being tested by confocal microscopy, single-cell traction force microscopy, or single-cell nanoindentation, among others.

Our results show that cells cultured in 3D environments decreased in size, increasing however, their biomechanical activity (i.e. cell-matrix anchorage forces). Interestingly, when increasing the stiffness of the artificial tumors, the phenotype known as "microcells" was discovered. These phenotypes correspond to extremely small cancer cells, characterized by the high degree of mutations, and their extreme pathogenicity. Microcells are traditionally generated by the use of several drugs (i.e. Paclitaxel), existing however, only a few reports showing the generation of these cells in vitro, and even less by the use of mechanically tunable polymer-based matrices.

Our results are then suggesting the crucial role played of mechanical stress in cancer progression, which can be effectively mimicked by the use of polymer-based Artificial Tumors, representing a simple, yet very reliable alternative to study cancer progression in vitro.