Introduction: Acral melanoma is a subtype of melanoma which develops in regions with high mechanical loads and low exposure to ultraviolet radiation, such as the soles of feet and palms. Compared to sun-exposed melanomas, they have higher genetic diversity, heterogeneity, and lead to higher mortality rates upon diagnosis due to their advanced tumor progression upon detection. Although some studies have demonstrated mechanical stress may play a role in pathogenesis, how mechanical stimuli contribute to acral melanoma and the mechanisms and pathways involved are not well understood. This study’s objective is to investigate how mechanical stimuli from the tumor microenvironment promote acral melanoma development, progression, and invasion. We aim to determine the impact of mechanical stimuli such as extracellular matrix architectural cues, mechanical compression, and spatial confinement on melanoma. Acral melanomas possess unevenly distributed irregular fibrillar patterns, and spatial architectures impart transcriptome heterogeneity in cancer tissues. We therefore hypothesize heterogeneous extracellular matrix architectures may differentially impact the phenotypic profiles of mechanically compressed or uncompressed melanoma cells.
Materials and
Methods: Novel biofabrication techniques are employed to generate hydrogels with heterogeneous collagen architectures comprising thickened, bundled collagen fibers, mimicking the complex architectures found in solid tumor microenvironments and in the dermis (Figure 1). Both human and mouse melanoma cells will be used for all studies, including the established mouse melanoma cell line B16F10 and human, patient-derived acral melanoma YUSEEP cells. Single melanoma cells and tumor spheroids will be embedded and cultured in these hydrogels with heterogenous architectures as well as regular collagen hydrogels and subjected to mechanical compression at 300 Pa pressures. Confocal microscopy will be performed on these samples, and quantitative biophysical data will be extracted from the resulting immunofluorescent images, including cell and nuclear morphologies, extracellular matrix remodeling, melanoma invasion profiles, and markers for proliferation, DNA damage, and DNA damage repair in response to these mechanical and architectural stimuli. To obtain mechanistic insights, melanoma cells will also be subjected to treatment with inhibitors for Rho kinase, actin, contractility, and matrix metalloproteinase, cultured in these hydrogels, and subjected to mechanical compression to determine if these inhibitors can rescue DNA damage. The mechanical load on single cells and spheroids in both regular collagen hydrogels and hydrogels with heterogeneous architectures will be computationally simulated with finite element modeling in COMSOL Multiphysics to further understand the stress distributions within the hydrogels. Single melanoma cells will also be subjected to pressure-driven flow and spatial constraints in a microfluidic platform to mechanically profile their deformations due to mechanical confinement through short and long constricted microchannels.
Results, Conclusions, and Discussions: Mechanically compressed B16F10 cells incur DNA damage (based on DNA damage marker γ-H2AX) in both bundled collagen and regular collagen, with bundled collagen architectures leading to lower DNA damage compared to regular collagen hydrogels (Figure 2). Since bundled collagen hydrogels densify less in response to compression compared to regular collagen fibers, the heterogenous architectures may provide a protective, caging effect against DNA damage. Melanoma cells in bundled collagen hydrogels exhibit more protrusions, with compressed bundled collagen samples leading to cells with longer protrusions, larger and less circular cells, and higher aspect ratios, indicating more cell spreading upon interactions with the architectural cues. Results have also indicated that YUSEEP tumor spheroids begin to demonstrate cell detachment and invasion within 72 h, with mechanical compression reducing invasion in both collagen and bundled collagen samples while simultaneously promoting cell proliferation (based on Ki-67 proliferation marker) on the tumor spheroid outer edges for compressed samples (Figure 3). Furthermore, tumor spheroids in compressed bundled collagen hydrogels exhibit more invasion than in compressed collagen hydrogels, indicating the architectural cues promote a more invasive phenotype. Additionally, both B16F10 and YUSEEP cells deform through short and long subnuclear sized constricted microchannels in response to 300 Pa of pressure gradient driven flow (Figure 4). The results from this study provide novel insights into the phenotypic profiles of melanoma cells in response to mechanical stimuli, the mechanism of acral melanoma progression, and may help elucidate new therapeutic targets as well as biophysical and molecular markers for diagnosing acral melanomas.
Acknowledgements (Optional): The authors gratefully acknowledge funding from NIH R35 Award GM142875 (MM), Yale Cancer Center Pilot Award (MM), and Yale Skin Cancer SPORE Award (MM). The human YUSEEP melanoma cells were a kind gift from Dr. Ruth Halaban’s Lab. The authors also thank members of the Mak Lab for their support.
