Multimodality Characterization of Cancer-Associated Fibroblasts in Tumor Microenvironment and Its Correlation With Ultrasound Shear Wave-Measured Tissue Stiffness in Localized Prostate Cancer

Abstract

Introduction: Growing evidence suggests that the tumor microenvironment (TME) represented by cellular and acellular components plays a key role in the multistep process of metastases and response to therapies. However, imaging and molecular characterization of the TME in prostate cancer (PCa) and its role in predicting aggressive tumor behavior and disease progression is largely unexplored. The study explores the PCa TME through the characterization of cancer-associated fibroblasts (CAFs) using both immunohistochemistry (IHC) and genomics approaches. This is then correlated with transrectal ultrasound shear wave elastography (USWE)-measured tissue stiffness.

Patients and methods: Thirty patients with clinically localized PCa undergoing radical prostatectomy for different risk categories of tumor (low, intermediate, and high) defined by Gleason score (GS) were prospectively recruited into this study. Prostatic tissue stiffness was measured using USWE prior to surgery. The CAFs within the TME were identified by IHC using a panel of six antibodies (FAP, SMAα, FSP1, CD36, PDGFRα, and PDGFRβ) as well as gene expression profiling using TempO-sequence analysis. Whether the pattern and degree of immunohistochemical positivity (measured by Quick score method) and expression of genes characterizing CAFs were correlated with USWE- and GS-measured tissue stiffnesses were tested using Spearman's rank correlation and Pearson correlation.

Results: There was a statistically significant correlation between GS of cancers, the pattern of staining for CAFs by immunohistochemical staining, and tissue stiffness measured in kPa using USWE (p < 0.001). Significant differences were also observed in immunohistochemical staining patterns between normal prostate and prostatic cancerous tissue. PDGFRβ and SMAα immunostaining scores increased linearly with increasing the USWE stiffness and the GS of PCa. There was a significant positive correlation between increasing tissue stiffness in tumor stroma and SMAα and PDGFRβ gene expression in the fibromuscular stroma (p < 0.001).

Conclusion: USWE-measured tissue stiffness correlates with increased SMAα and PDGFRβ expressing CAFs and PCa GSs. This mechanistic correlation could be used for predicting the upgrading of GS from biopsies to radical surgery and response to novel treatments.

Keywords: cancer-associated fibroblasts; immunohistochemistry; prostate cancer; stiffness; tumor microenvironment.

Figure 1

Images obtained from a 72-year-old man. (A) USWE image; the average mean of the stiffness was 143.6 kPa. (B) Histopathology image of the prostate cancer; the final diagnosis was prostate cancer with Gleason score 4 + 5. (C) IHC of SMAα with dilution (1/50 + linker) in prostate cancer tissue. 400× magnification. (D) The relationship between the Gleason score and the gene expression of the SMAα. (E) The relationship between the Gleason score and the IHC of the SMAα.

Figure 2

Schematic of TempO-Seq assay protocol.

Figure 3

IHC using six antibodies to stain CAFs in different Gleason scores of prostate cancer tissues. Images for (A) FAP with dilution (1/100), (B) SMAα with dilution (1/50 + linker), (C) FSP1 with dilution (1/5,000), (D) CD36 with dilution (1/250), (E) PDGFRα with dilution (1/100 + linker), and (F) PDGFRβ with dilution (1/100). (1) 3 + 3 GS, (2) 3 + 4 GS, (3) 4 + 3 GS, (4) 3 + 5 GS, and (5) 4 + 5 GS. 400× magnification.

Figure 4

The scatter plots show the relationship between the stiffness of the Ultrasound shear wave elastography and the score of immunohistochemistry intensity and density in (A) FSP1, (B) SMAα, (C) FAP, (D) PDGFRα, and (E) PDGFRβ. (B) [rs (28) = 0.73, p < 0.0010], and (E) [rs (28) = 0.73, p < 0.001], the correlation of the other genes were not statistically significant.

Figure 5

The scatter plots show the relationship between the Gleason score and the gene expression of the SMAα and PDGFRβ [r _s(28) = 0.58, p = 0.001, and r _s(28) = 0.56, p = 0.001], respectively.

Figure 6

Representative figure showing a change in histological GS following radical surgery and corresponding expression of CAFs and genomic analysis. Note tissue stiffness measured in kPa using ultrasound shear wave elastography.