The relationship between the tumor microenvironment of hepatocellular carcinoma - including cancer-associated fibroblasts and tumor-associated macrophages - and apparent diffusion coefficient

Abstract

The tumor microenvironment is critical for the acquisition of tumor malignancy in various cancer types. The objectives of this study were to investigate whether the levels of cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) reflect the prognosis of patients with hepatocellular carcinoma (HCC) after hepatectomy (Hx) and to determine whether the apparent diffusion coefficient (ADC) from diffusion-weighted imaging (DWI) reflects CAF and TAM expression. The study cohort comprised 109 patients who underwent initial curative resection for HCC. Alpha smooth muscle actin (αSMA) was selected as a CAF marker and CD204 as a TAM marker. Protein expression was immunohistochemically evaluated in the intratumoral regions of resected specimens. Clinicopathological factors, including the long-term prognosis after Hx, were investigated between αSMA-negative and -positive tumors and between CD204-negative and -positive tumors. The correlation between CAF/TAM marker expression and the calculated minimum ADC using DWI was also evaluated. αSMA-positive expression was correlated with tumor number, invasive growth pattern, and advanced stage. CD204-positive expression was correlated with the presence of venous invasion. Both αSMA-positive expression and CD204-positive expression were significant prognostic factors in the univariate analysis of overall survival and disease-free survival. αSMA/CD204 double positivity was associated with an extremely poor prognosis after Hx and was a significant independent prognostic factor for overall survival (P=0.02, hazard ratio: 3.27). Patients with double positivity also showed a significantly higher ADC^low rate (83%). In conclusion, expression of both CAF and TAM markers reflected a poor prognosis after Hx. Furthermore, the preoperative ADC could be a clinical surrogate marker in the tumor microenvironment in patients with HCC.

Keywords: Tumor microenvironment; apparent diffusion coefficient; cancer-associated fibroblast; hepatocellular carcinoma; tumor-associated macrophage.

Figure 1

Immunohistochemistry of αSMA. αSMA expression scores were assessed based on both staining area (0, 0%-50%; 1, ≥50% positive area) and intensity (0, weak; 1, mild; 2, strong). A. “Area score 0” and “staining score 0 or 1” were considered to indicate negativity. B. “Area score 1” or “area score 0” and “staining score 2” were considered to indicate positivity.

Figure 2

Immunohistochemistry of CD204. For the CD204 staining, positive cells were counted, and “mean positive cells <90 cells/HPF” were considered to indicate negativity (A), while “mean positive cells ≥90 cells/HPF” were considered to indicate positivity (B).

Figure 3

ADC calculation with minimum value. Synapse Vincent can show mean, minimum, and maximum values from free-form green outlines automatically. Minimum ADC values were used because our previously reports showed minimum ADC value predicted patients’ prognosis.

Figure 4

Long-term prognosis. A. CAF marker. CAF marker positive patients showed poor prognosis after Hx in both OS (P=0.02) and DFS (P=0.04). B. TAM marker. TAM marker positive patients showed poor prognosis after Hx in both OS (P=0.04) and DFS (P=0.06). C. Correlation between CAF and TAM expression. The expression of CAF and TAM marker had positive correlation (P=0.04). D. Prognostic stratification with both CAF and TAM markers. When patients were divided into a CAF/TAM double-negative group, a CAF-positive or TAM-positive group, and a CAF/TAM double-negative group, both OS and DFS were clearly stratified by these two markers (P<0.01).

Figure 5

ADC value as clinical surrogate marker in TME. ADC minimum values of CAF/TAM double positive group showed lower value than other groups (Double negative group; 1.06±0.05, Positive and negative group; 1.02±0.09, Double positive group; 0.82±0.09, P=0.06; Double negative vs. Double positive group). Under the criteria of cut-off value; 0.74×10^-3 mm²/s, CAF/TAM double positive group showed significantly higher ADC^low rate (Double negative group; 13%, Positive and negative group; 19%, Double positive group; 83%, P=0.01).