Variations in tumor biology from patient to patient combined with the low overall survival rate of hepatocellular carcinoma (HCC) present significant clinical challenges. During the progression of ...chronic liver diseases from inflammation to the development of HCC, microenvironmental properties, including tissue stiffness and oxygen concentration, change over time. This can potentially impact drug metabolism and subsequent therapy response to commonly utilized therapeutics, such as doxorubicin, multi-kinase inhibitors (e.g., sorafenib), and other drugs, including immunotherapies. In this study, we utilized four common HCC cell lines embedded in 3D collagen type-I gels of varying stiffnesses to mimic normal and cirrhotic livers with environmental oxygen regulation to quantify the impact of these microenvironmental factors on HCC chemoresistance. In general, we found that HCC cells with higher baseline levels of cytochrome p450-3A4 (CYP3A4) enzyme expression, HepG2 and C3Asub28, exhibited a cirrhosis-dependent increase in doxorubicin chemoresistance. Under the same conditions, HCC cell lines with lower CYP3A4 expression, HuH-7 and Hep3B2, showed a decrease in doxorubicin chemoresistance in response to an increase in microenvironmental stiffness. This differential therapeutic response was correlated with the regulation of CYP3A4 expression levels under the influence of stiffness and oxygen variation. In all tested HCC cell lines, the addition of sorafenib lowered the required doxorubicin dose to induce significant levels of cell death, demonstrating its potential to help reduce systemic doxorubicin toxicity when used in combination. These results suggest that patient-specific tumor microenvironmental factors, including tissue stiffness, hypoxia, and CYP3A4 activity levels, may need to be considered for more effective use of chemotherapeutics in HCC patients.
Understanding the effects of inflammation and cirrhosis on the regulation of drug metabolism during the progression of hepatocellular carcinoma (HCC) is critical for developing patient-specific ...treatment strategies. In this work, we created novel three-dimensional vascularized HCC-on-a-chips (HCCoC), composed of HCC, endothelial, stellate, and Kupffer cells tuned to mimic normal or cirrhotic liver stiffness. HCC inflammation was controlled by tuning Kupffer macrophage numbers, and the impact of cytochrome P450-3A4 (CYP3A4) was investigated by culturing HepG2 HCC cells transfected with CYP3A4 to upregulate expression from baseline. This model allowed for the simulation of chemotherapeutic delivery methods such as intravenous injection and transcatheter arterial chemoembolization (TACE). We showed that upregulation of metabolic activity, incorporation of cirrhosis and inflammation, increase vascular permeability due to upregulated inflammatory cytokines leading to significant variability in chemotherapeutic treatment efficacy. Specifically, we show that further modulation of CYP3A4 activity of HCC cells by TACE delivery of doxorubicin provides an additional improvement to treatment response and reduces chemotherapy-associated endothelial porosity increase. The HCCoCs were shown to have utility in uncovering the impact of the tumor microenvironment (TME) during cancer progression on vascular properties, tumor response to therapeutics, and drug delivery strategies.
Most platforms used for the molecular reconstruction of the tumor-immune microenvironment (TIME) of a solid tumor fail to explore the spatial context of the three-dimensional (3D) space of the tumor ...at a single-cell resolution, and thus lack information about cell-cell or cell-extracellular matrix (ECM) interactions. To address this issue, a pipeline which integrated multiplex spatially resolved multi-omics platforms was developed to identify crosstalk signaling networks among various cell types and the ECM in the 3D TIME of two FFPE (formalin-fixed paraffin embedded) gynecologic tumor samples. These platforms include non-targeted mass spectrometry imaging (glycans, metabolites, and peptides) and Stereo-seq (spatial transcriptomics) and targeted seqIF (IHC proteomics). The spatially resolved imaging data in a two- and three-dimensional space demonstrated various cellular neighborhoods in both samples. The collection of spatially resolved analytes in a voxel (3D pixel) across serial sections of the tissue was also demonstrated. Data collected from this analytical pipeline were used to construct spatial 3D maps with single-cell resolution, which revealed cell identity, activation, and energized status. These maps will provide not only insights into the molecular basis of spatial cell heterogeneity in the TIME, but also novel predictive biomarkers and therapeutic targets, which can improve patient survival rates.
Primary liver cancer, or hepatocellular carcinoma (HCC), is a major worldwide cause of death from carcinoma. Most patients are not candidates for surgery and medical therapies, including new ...immunotherapies, have not shown major improvements since the modest benefit seen with the introduction of sorafenib over a decade ago. Locoregional therapies for intermediate stage disease are not curative but provide some benefit. However, upon close scrutiny, there is still residual disease in most cases. We review the current status for treatment of intermediate stage disease, summarize the literature on correlative histopathology, and discuss emerging methods at micro-, nano-, and pico-scales to improve therapy. These include transarterial hyperthermia methods and thermoembolization, along with microfluidics model systems and new applications of mass spectrometry imaging for label-free analysis of pharmacokinetics and pharmacodynamics.
Patient‐Specific TreatmentIn article 2200403, Alican Özkan, and co‐workers created a novel vascularized hepatocellular carcinoma on a chip (HCCoC) composed of tunable collagen and four main ...liver‐specific cells, which serves as a translational testing tool. HCCoC is used to assess vascular transport regulation and efficacy of therapeutics delivered with embolization and systematic methods during the cancer progression.
There are three main outcomes of Mycobacterium tuberculosis infection: clearance, dissemination, and containment - in which the immune system physically isolates the invading microbes in lesions ...called granulomas. These structures are a hallmark of the disease and play an important role in the progression of infection. However, current in vitro and in vivo methods are ill adapted for spatial and temporal quantification of host-pathogen dynamics, which are necessary for the development of granulomas. We have developed an integrated 3D in vitro and computational platform with long-term time-lapse confocal imaging to provide a semi-automatic analysis of host-pathogen interaction data. Through exploratory data analysis, we conduct a preliminary investigation of how the intracellular bacterial load of macrophages can impact cellular spatiotemporal dynamics during Mycobacterium infection.
Host response to Mycobacterium tuberculosis (Mtb) is distinctive in the use of a spatial immunological response to limit the progression of infection. This results in the formation granulomas, ...aggregations of immune cells that isolate invading microbes, a hallmark of the adaptive immune response to Mtb infection. Traditional in vivo studies have investigated the mature granuloma, but relatively fewer studies investigate how these structures form during the early stages of infection nor how spatial organization impacts control, resolution, or dissemination of the bacterium. Research has shown that initial aggregation of macrophages during innate immune response influences the progression of disease and formation of granulomas during adaptive immunity. However, current experimental methods for studying cellular interactions during early stages of infection are ill adapted for concurrent spatial and temporal quantification of host-pathogen dynamics, which is necessary for a quantitative understanding of the innate spatial immune response to Mtb and to inform the development of accurate computational models of tuberculosis disease. To address this, we developed a three-dimensional (3D) ex vivo model of mycobacterium infected macrophages cultured in reconstituted basement membrane and characterized the structural impact of 3D structure on infection dynamics in comparison to standard two-dimensional (2D) infection models. We quantified temporal immune response using standard biological sampling methodologies and long-term time-lapse confocal imaging to quantify the early spatiotemporal dynamics of macrophage response to mycobacterium infection. Our studies using Mycobacterium smegmatis indicate that the 3D environment induces a shift in dynamics. In 3D we see significantly higher cellular velocities in infected conditions as compared to control non-infected conditions, whereas the converse occurs in 2D. This may impact computational models that utilize 2D assumptions. We developed a data analysis pipeline to quantify macrophage state with respect to infection and cellular microenvironment. Results show non-infected and non-active macrophages within infected environments present dynamics comparable to controls, while infected and activated macrophages exhibit comparable spatiotemporal dynamics in 2D and 3D. Using the more virulent Mycobacterium bovis BCG, we observe a greater distinction between control and infected conditions and preliminary evidence of a more distinct 3D immune response resulting in increased cell death and extracellular bacteria.
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