12th Annual Symposium
Physics of Cancer
Aug 30 - Sept 1, 2021
|PoC - Physics of Cancer - Annual Symposium|
Lipid-droplet mediated nuclear deformation occurs independently of cytoskeletal forces in hepatocytes
Introduction: Hepatocellular carcinoma (HCC) is a deadly primary liver cancer, resulting in ~800,000 deaths globally per year . The mechanisms of HCC development and progression are not well characterized, although ~80% of HCC occurs in stiff cirrhotic livers, and increased tissue stiffness correlates to poorer clinical outcomes [2,3], suggesting that mechanical stress may contribute to the initiation and progression of the disease. However, HCC can occur in soft livers with non-alcoholic fatty liver disease (NAFLD) with minimal amounts of fibrosis. We hypothesized that lipid droplet accumulation in NAFLD acts as an intracellular mechanical stress in hepatocytes, initiating oncogenesis at lower tissue stiffness by disrupting the cell cytoskeleton, and directly deforming the nucleus. Computational models of lipid droplet-mediated nuclear deformation suggested that, due to the relative stiffness of the cell nucleus, active cytoskeletal forces may be necessary to induce large scale nuclear deformations, raising the possibility that lipid-loading and substrate stiffness may work synergistically to induce nuclear deformation.
Materials and Methods: Primary human hepatocytes or isolated rat hepatocytes were cultured on glass and on 500 Pa and 10 kPa polyacrylamide gels, all coated with 1 mg/mL type 1 collagen. After 48 hrs cells were switched to serum-free media supplemented with 400 nM oleate (conjugated to bovine serum albumin) and incubated an additional 48 hrs. To determine whether lipid droplets can directly deform the nucleus, we also inhibited actin polymerization, microtubule polymerization and cell contraction (using 5 uM latrunculin, 10 uM nocodazole, or 5 uM blebbistatin, respectively) for 4 hrs after lipid loading. Cells were fixed and stained using DAPI for nuclei and BODIPY for neutral lipids, and nuclear shape and chromatin organization were imaged by confocal microscopy. HNF-4alpha was stained to determine whether deformation correlates to hepatocyte dedifferentiation. To compare between groups staining intensity was quantified in ImageJ and nuclear deformation was quantified using a specially developed MATLAB program.
Results and Discussion: Primary human hepatocytes readily process lipid droplets, leading to accumulation in the cell cytoplasm. Lipid accumulation is increased on softer substrates with more dense packing of lipid droplets and the nuclei of lipid-loaded cells have lower volume and spread area than controls. Cell volume of lipid loaded cells remains unchanged, however, indicating that hepatocytes are not expanding to accommodate the lipid volume. Lipid droplets visibly deform and indent the nucleus on all stiffness substrates, and when quantified significantly more so in oleate-treated cells than controls. However, the nuclei of oleate-treated hepatocytes on stiff substrates were taller than untreated cells, suggesting that lipid droplets may resist compression in the YZ plane. Experiments inhibiting actin polymerization, microtubule polymerization and myosin-mediated cell contraction showed that lipid droplets indent the nucleus even in the absence of an intact cytoskeletal network, suggesting that the lipid droplets themselves directly exert mechanical stress on the nucleus. The nuclei of oleate-treated hepatocytes were essentially insensitive to cytoskeletal treatment both in radial indentation by droplets and nuclear height, while control cells exhibited altered nuclear morphology as expected. Preliminary experiments indicate that HNF-4alpha may also be reduced in oleate-treated cells, suggesting that nuclear deformation by droplets may lead to hepatocyte dedifferentiation in a similar manner as does increased stiffness.
Conclusions: Lipid-loading of hepatocytes leads to compression and deformation of the nucleus in the XY plane, although lipid droplets appear to resist compression in the YZ plane. Inhibition of the cytoskeleton through various drug treatments has little impact on hepatocyte morphology in oleate-treated cells, suggesting that lipid droplets themselves directly indent the nucleus. Taken together, these results suggest that the intracellular lipid droplets in hepatocytes seen in NAFLD cause nuclear stress and could lead mechanosensitive signaling and differentiation in softer environments.
Acknowledgements: We acknowledge the funding support of the NSF Graduate Research Fellowship, PSOC@Penn (NIH U54 CA193417), the NIDDK Molecular Pathology & Imaging Core (NIH P30 DK050306), and the NSF Center for Engineering MechanoBiology (CMMI:15-48571).
References:  Rawla P, Contemp Oncol, 2018; 22(3):141-150  Rahib L, Cancer Res, 2014; 74:2913-21  Tatsumi A, Hepatol Res, 2015; 21:214-19