Computational/experimental evaluation of liver metastasis post hepatic injury: interactions with macrophages and transitional ECM

Abstract

The complex interactions between subclinical changes to hepatic extracellular matrix (ECM) in response to injury and tumor-associated macrophage microenvironmental cues facilitating metastatic cell seeding remain poorly understood. This study implements a combined computational modeling and experimental approach to evaluate tumor growth following hepatic injury, focusing on ECM remodeling and interactions with local macrophages. Experiments were performed to determine ECM density and macrophage-associated cytokine levels. Effects of ECM remodeling along with macrophage polarization on tumor growth were evaluated via computational modeling. For primary or metastatic cells in co-culture with macrophages, TNF-α levels were 5× higher with M1 vs. M2 macrophages. Metastatic cell co-culture exhibited 10× higher TNF-α induction than with primary tumor cells. Although TGFβ1 induction was similar between both co-cultures, levels were slightly higher with primary cells in the presence of M1. Simulated metastatic tumors exhibited decreased growth compared to primary tumors, due to high local M1-induced cytotoxicity, even in a highly vascularized microenvironment. Experimental analysis combined with computational modeling may provide insight into interactions between ECM remodeling, macrophage polarization, and liver tumor growth.

Figure 1

Transwell assays. (A) Relative migration of each tumor cell line on each ECM substrate, normalized to the respective “negative” control group of uncoated transwell membrane with no ECM (nECM) substrate coating. Five separate fields per slide were counted and averaged. Serum-free (SF) media controls were used to establish baseline migration with no chemoattractant in receiver well. Groups on the horizontal axis relate to growth conditions; within each group there is a representative bar for each cell line-ECM combination. Error bars represent the standard error of the mean, while horizontal bars highlight significant differences among pairwise comparisons. Primary tumor cells on transitional ECM (tECM) were included as a baseline for comparison, noting that they may not represent a biologically relevant case. Cells grown on FBS-supplemented control ECM substrate (“FBS cECM”) showed significant differences in relative migration between primary and metastatic cells (P < 0.001). Cells in serum-free tECM substrate (“SF tECM”) also showed significant differences in relative migration (P < 0.05). In supplemented media, relative migration of primary cells was shown to be different with respect to the ECM substrate (P < 0.001). (B) Macrophage-tumor cells indirect co-culture assay. TNF-α levels indicated a significant difference between the cases with M1-activated macrophages and the cases with naïve (“control”) or M2 macrophages (P ≤ 0.05). (C) Control transwells without tumor cells in the insert showed no differences in TGFβ1 levels across the treatment groups. NC: No tumor cells present; PT: with primary tumor cells; MT: with metastatic tumor cells.

Figure 2

ECM-dependent simulations (naïve macrophage populations only). Simulated tumor growth at 13 d with ECM degradation/production ratio calibrated to simulate murine hepatic control ECM (cECM) and the transitional ECM (tECM) induced by alcohol exposure. The output matrix shows primary and metastatic tumors in the top and bottom rows, respectively; cECM and tECM simulations are shown in the first and second columns, respectively. Each 3 × 3 grid, comprised of nine 4 mm² panels, shows the tumor and vessels (brown lines) in the top left corner, with proliferating regions in red, quiescent hypoxic regions in blue, and necrosis in brown. Both primary tumor simulations in the top row have larger areas of necrosis than the metastatic tumor simulations in the bottom row, which have mostly hypoxic cores. The vasculature grid for metastatic cells was calibrated to be denser, to recapitulate the high density of liver vasculature. In the middle and right corners of the top row, the simulated macrophage chemoattractants, TNF-α and TGFβ1, are shown in heat map as they are secreted in the tumor microenvironment. The middle leftmost panel of each grid shows the naïve macrophages extravasated from the surrounding vasculature. The middle center and rightmost panel of each grid show the density of Type 1 (M1) and Type 2 (M2) macrophages, respectively, polarized in the tumor microenvironment. In these simulations, the polarization is turned off in order to only evaluate the effect of the ECM variations. The bottom middle and leftmost panels of each grid show the simulated tumor oxygenation and tumor angiogenic factors (e.g., VEGF), respectively.

Figure 3

ECM-dependent simulations (with polarized macrophage populations). Simulated tumor growth at 13 d with macrophage polarization turned on (same grid panel descriptions as in Fig. 2). Both macrophage phenotypes appear to have penetrated the tumor mass. Metastatic tumors have a higher infiltration of M1 subtypes, which results in growth restriction relative to the primary cases.

Figure 4

Simulated tumor size. Average tumor radius by 13 d. With polarized macrophages present, metastatic tumors had a smaller tumor radius than primary tumors. “Control” simulation tumor radii (with naïve macrophages only) were comparable for either ECM construct. Error bars represent standard deviation with n = 3; asterisk denotes significance calculated via student t-test, p-value < 0.05.

Figure 5

Simulated macrophage population fractions. Time evolution of macrophage subtype populations for (A) primary tumor on control ECM, (B) metastatic tumor on control ECM, (C) primary tumor on transitional ECM, and (D) metastatic tumor on transitional ECM. Primary tumor simulations of either ECM construct exhibited more balanced proportion of M1:M2 macrophage subtypes than metastatic tumors; on either ECM construct, M2 macrophages were more prominent than M1 for primary tumors. In contrast, M1 macrophages dominated for the metastatic tumors.