Abstract
The routine study of human malaria liver-stage biology in vitro is hampered by low infection efficiency of human hepatocellular carcinoma (HCC) lines (<0.1%), poor understanding of steady-state HCC biology, and lack of appropriate tools for trace sample analysis. HC-04 is the only HCC that supports complete development of human malaria parasites. We hypothesized that HCCs are in various intermediate stages of the epithelial-mesenchymal transition (EMT) and HC-04s retain epithelial characteristics that permit infection. We developed a facile analytical approach to test this hypothesis viz. the HC-04 response to hepatocyte growth factor (HGF). We used online two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) to quantify protein expression profiles in HC-04 pre-/post-HGF treatment and validated these results by RT-qPCR and microscopy. We successfully increased protein identification efficiency over offline-2D methods by 12-fold, using less sample material, allowing robust protein quantification. We observed expected up-regulation and down-regulation of EMT protein markers in response to HGF, but also unexpected cellular responses. We also observed that HC-04 is generally more susceptible to HGF-mediated signaling than what was observed for HepG2, a widely used, but poor malaria liver stage-HCC model. Our analytical approach to understanding the basic biology of HC-04 helps us understand the factors that may influence its utility as a model for malaria liver-stage development. We observed that HC-04 treatment with HGF prior to the addition of Plasmodium falciparum sporozoites did not facilitate cell invasion, which suggests unlinking the effect of HGF on malaria liver stage development from hepatocyte invasion. Finally, our 2D-LC-MS/MS approach and broadly applicable experimental strategy should prove useful in the analysis of various hepatocyte-pathogen interactions, tumor progression, and early disease events.
Highlights
We hypothesized that the acute response of HC-04 to Hepatocyte Growth Factor (HGF) would be an indication of its relative stage along the epithelial to mesenchymal transition (EMT) spectrum, and that the specific regulation of the widely implicated EMT cellular markers can help us identify the possible facilitators of LS invasion and development
It is interesting to speculate that up-regulation of PBGD in HC-04 results in increased enzyme activity, a phenomenon that has been described in C6 glioma cells, wherein overexpression of PBGD resulted in G1 cell cycle attenuation, reduction in synthesis of Vimentin and subsequent transformation of C6 cells into a more astrocyte-like morphology [35]
It should be noted that there is growing evidence that expression of the implicated EMT markers are extremely heterogeneous in hepatocellular carcinoma (HCC) [36, 37], and expression is dependent on size and location within tumors, and that HCC cell lines may not be an exception [36]
Summary
Cell Culture—HC-04 and HepG2 cells were grown in Iscove’s Modified Dulbecco’s Medium (IMDM) (Invitrogen, Carlsbad, CA) supplemented with 5% heat-inactivated fetal bovine serum (Corning Life Sciences, Tewksbury, MA), and Penicillin/streptomycin (Invitrogen) in a humidified chamber at 5% CO2. Cells were fixed and stained following our standard protocol (see Supplemental Methods). After a standard TriZOL RNA extraction protocol was followed, samples were DNase treated and further purified using an RNeasy kit (Qiagen, Gaithersburg, MD). The process of calculating normalized spectral counts is as follows: (a) Scaffold takes the sum of all the Total Spectrum Counts for each MS sample; (b)The sums are scaled to the same level; and (c) Scaffold applies the scaling factor for each sample to each protein group to produce an output with a normalized quantitative value. Plasmodium falciparum Sporozoite HC-04 Invasion Assay— HC-04 cells were grown in equal volumes DMEM (Invitrogen) and Ham’s F-12 nutrient mix (Invitrogen) supplemented with 10% heatinactivated fetal bovine serum (Corning Life Sciences; Tewksbury, MA), 15 mM HEPES (Sigma Aldrich), 1.5g/L sodium bicarbonate (Sigma Aldrich), and Penicillin/streptomycin (Invitrogen; Carlsbad, CA) in a humidified chamber at 5% CO2. The percentage of HC-04 cells infected was calculated by division of the number of internalized sporozoites by the number of HC-04 cells (between 2000 – 4000 cells), followed by multiplication by 100
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