The prometastatic microenvironment of the liver

Abstract

The liver is a major metastasis-susceptible site and majority of patients with hepatic metastasis die from the disease in the absence of efficient treatments. The intrahepatic circulation and microvascular arrest of cancer cells trigger a local inflammatory reaction leading to cancer cell apoptosis and cytotoxicity via oxidative stress mediators (mainly nitric oxide and hydrogen peroxide) and hepatic natural killer cells. However, certain cancer cells that resist or even deactivate these anti-tumoral defense mechanisms still can adhere to endothelial cells of the hepatic microvasculature through proinflammatory cytokine-mediated mechanisms. During their temporary residence, some of these cancer cells ignore growth-inhibitory factors while respond to proliferation-stimulating factors released from tumor-activated hepatocytes and sinusoidal cells. This leads to avascular micrometastasis generation in periportal areas of hepatic lobules. Hepatocytes and myofibroblasts derived from portal tracts and activated hepatic stellate cells are next recruited into some of these avascular micrometastases. These create a private microenvironment that supports their development through the specific release of both proangiogenic factors and cancer cell invasion- and proliferation-stimulating factors. Moreover, both soluble factors from tumor-activated hepatocytes and myofibroblasts also contribute to the regulation of metastatic cancer cell genes. Therefore, the liver offers a prometastatic microenvironment to circulating cancer cells that supports metastasis development. The ability to resist anti-tumor hepatic defense and to take advantage of hepatic cell-derived factors are key phenotypic properties of liver-metastasizing cancer cells. Knowledge on hepatic metastasis regulation by microenvironment opens multiple opportunities for metastasis inhibition at both subclinical and advanced stages. In addition, together with metastasis-related gene profiles revealing the existence of liver metastasis potential in primary tumors, new biomarkers on the prometastatic microenvironment of the liver may be helpful for the individual assessment of hepatic metastasis risk in cancer patients.

Fig. 1

Scanning electron microscopic image on the hepatic lobule. The hepatic lobule has the form of a polyhedral prism that in this picture has been sectioned horizontally. At the lobule corners, portal tracts are situated containing terminal portal veins (TPV), perilobular arteries, lymphatic vessels, nerve fibers and bile ducts (not visible). TPV surround the lobule and are also connected to sinusoids through occasional gates serving for the intralobular access of blood (arrows). Hepatic parenchymal cells—hepatocytes (H)—are organized in plates radially arranged around the centrilobular vein (CLV) located in the center of the hepatic lobule. Sinusoids (S) form a microvasculature among hepatocytes within the hepatic lobules. They form an anastomotic network in the periportal area, while are straits in the centrilobular area around the central vein. Blood passes through openings in the TPV into sinusoids and circulates among hepatocytes to be collected by the CLV. There, blood continues to the interlobular veins and, then, into collecting veins draining finally into the hepatic veins leaving the liver through the suprahepatic vein (not shown). Bar: 50 μm

Fig. 2

Scanning electron microscopic images on the intrahepatic pathway of metastatic cancer cells. a Cross section of a 9-μm in diameter hepatic sinusoid (S), lined by the fenestrated sinusoidal endothelium (E), and surrounded by hepatocytes (H), bar: 5 μm. b An intrasinusoidal Kupffer cell occupying the sinusoidal lumen and connected to the endothelial wall by filopodia and a long citoplasmic prolongation (arrows), bar: 5 μm. c Fenestrated surface of the hepatic sinusoidal endothelium. Under physiological conditions, endothelial fenestrae are transcellular structures of 100–150 nm in diameter that cluster forming highly filtrating microdomains named sieve plates, bar: 1 μm. d Mouse liver tissue on the fifth day post-intrasplenic injection of Lewis lung carcinoma cells. Circulating cancer cells first interact with non-fenestrated endothelial cells and adhered leukocytes and macrophages at perilobular terminal portal veins (TPV). Pre-sinusoidal gates for intralobular access of cancer cells (S). Intrasinusoidal retention of cancer cells (arrows) within the periportal area of the hepatic lobule. Surrounding hepatocytes (H) and endothelial cells (E) lining sinusoids (S). Bar: 10 μm

Fig. 3

Confocal microscopy on experimental cancer cell entry and residence in the hepatic microvasculature. Carboxyfluorescein-labeled CT26 murine colorectal carcinoma cells were intrasplenically injected in syngeneic mice and their livers were perfused on the 24th and 48th hour with fluorescence-labeled wheat germ agglutinin (WGA), as previously described [7], for the specific staining of hepatic microvascular walls (cancer cells had not WGA-binding sites. a Retention of a cancer cell (in green) in the lumen of a hepatic sinusoid (in red). Notice that the body of the cancer cell completely occupied the sinusoidal lumen preventing endothelial cell labeling with the perfused lectin beyond this point. b Cancer cell adhesion to the endothelial wall of a periportal sinusoidal segment. c Onset of cancer cell proliferation on the 48th after injection. Notice that cancer cell clump did not alter microvascular staining in the area, suggesting the extravascular position of cancer cells. Terminal portal vein (TPV); sinusoid (S); cancer cells (arrows). Bar: 20 μm

Fig. 4

Tumor-induced proinflammatory factors regulate melanoma cell adhesion to hepatic sinusoidal endothelium prior to metastasis formation

Fig. 5

Light microscopic image of hepatic tissue. a Parenchymal cell heterogeneity across the hepatic lobule, as shown by immunohistochemical staining of nerve growth factor-expressing hepatocytes. Bar: 100 μm. b Periportal micrometastases (arrows) from intrasplenically-injected B16F10 melanoma cells. Terminal portal veins (TPV) and centrilobular veins (CLV). Bar: 100 μm

Fig. 6

a Two mechanisms are proposed for the hepatic stellate cell activation by tumor-derived factors: Indirect, via tumor-activated hepatic sinusoidal endothelial cells; direct, via transendothelial cell diffusion of tumor-derived hepatic stellate cell-stimulating factors. b Intratumoral recruitment of hepatic stellate cells (red stained) at the intralobular micrometastasis phase of the experimental colonization of intrasplenically-injected C26 colon carcinoma cells. Immunohistochemical detection of smooth muscle-alpha actin reveals that intrametastatic, but not extrametastatic, hepatic stellate cells become myofibroblasts by tumor-derived paracrine/juxtacrine factors prior to angiogenesis occurrence. Terminal portal veins (TPV) surrounded by portal tract fibroblasts and smooth muscle cells (in red). Bar: 50 μm. c Panlobular C26 colon carcinoma micrometastasis containing a dense population of tumor-activated myofibroblasts as revealed by immunohistochemical detection of smooth muscle alpha-actin (stained in brown). Bar: 100 μm. d High-magnification picture on smooth muscle alpha-actin-expressing stromal myofibroblasts (MF) in an established hepatic metastasis from a colorectal carcinoma patient. Tumor cells (TC). Bar: 15 μm

Fig. 7

Low (a) and high (b) magnification pictures on the intratumoral recruitment of hepatocytes in a C26 colon carcinoma intralobular micrometastasis (MET). Tumor-activated perimetastatic and intrametastatic hepatocytes (in brown) were immunohistochemicaly revealed by anti-mouse nerve growth factor (NGF) antibody. Only cholangiocytes within the perilobular bile ducts and periportal hepatocytes express NGF under normal physiological conditions. However, tumor microenvironmental factors induced hepatocyte expression of NGF. Bar: 100 μm (a) and 10 μm (b)

Fig. 8

Immunohistochemical detection of CD31-expressing angiogenic endothelial cells in portal-type (pushing growth pattern) (a) and sinusoidal-type (replacement growth pattern) (c) hepatic metastases from intrasplenically-injected head and neck squamous cell murine PAN-LY2 carcinoma cells. Immunohistochemical staining for smooth muscle alpha actin expression of serial tissue sections from the same livers (b and d). Notice the co-localization of CD31 and smooth muscle alpha actin expressing cells in both kinds of hepatic metastases. Bar: 150 μm. e High-magnification confocal microscopic image on intrametastatic neo-angiogenic vessels. CD31-expressing endothelial cells (green-stained) were surrounded by smooth muscle alpha actin-expressing vascular coverage cells (red stained). Bar: 20 μm

Fig. 9

a Avascular micrometastasis (arrows) developed in the sinusoidal domain of an hepatic lobule, surrounded by tumor-activated hepatic stellate cells expressing smooth muscle alpha actin (red stained cells). Terminal portal venule (TPV). Hepatocytes (H). Bar: 25 μm. b Sinusoidal-type hepatic micrometastasis (MET) at the angiogenic phase, containing a dense network of sinusoidal neovessels, as revealed by reticulin stain according to Gordon–Sweets silver impregnation technique. Recruited microvessels form concentric interconnections. Liver architecture is not disturbed, and cancer cells co-opt the supportive fibrilar network of the sinusoids. Bar: 100 μm. c Avascular micrometastasis (arrows) developed in close proximity to a terminal portal vein (TPV) and surrounded by portal tract-derived cells expressing smooth muscle alpha actin (red stained cells). Bar: 25 μm. d Portal-type micrometastasis (MET) at the angiogenic phase. Here, the reticulin network supporting intratumoral angiogenesis is not conserved. Desmoplastic stroma surrounds and traverses metastasis, facilitating invasion of vascular-type angiogenic vessels. Necrotic areas frequently develop in this metastasis type. Bar: 100 μm