A distinct repertoire of cancer-associated fibroblasts is enriched in cribriform prostate cancer

Abstract

Outcomes for men with localized prostate cancer vary widely, with some men effectively managed without treatment on active surveillance, while other men rapidly progress to metastatic disease despite curative-intent therapies. One of the strongest prognostic indicators of outcome is grade groups based on the Gleason grading system. Gleason grade 4 prostate cancer with cribriform morphology is associated with adverse outcomes and can be utilized clinically to improve risk stratification. The underpinnings of disease aggressiveness associated with cribriform architecture are not fully understood. Most studies have focused on genetic and molecular alterations in cribriform tumor cells; however, less is known about the tumor microenvironment in cribriform prostate cancer. Cancer-associated fibroblasts (CAFs) are a heterogeneous population of fibroblasts in the tumor microenvironment that impact cancer aggressiveness. The overall goal of this study was to determine if cribriform prostate cancers are associated with a unique repertoire of CAFs. Radical prostatectomy whole-tissue sections were analyzed for the expression of fibroblast markers (ASPN in combination with FAP, THY1, ENG, NT5E, TNC, and PDGFRβ) in stroma adjacent to benign glands and in Gleason grade 3, Gleason grade 4 cribriform, and Gleason grade 4 noncribriform prostate cancer by RNAscope®. Halo® Software was used to quantify percent positive stromal cells and expression per positive cell. The fibroblast subtypes enriched in prostate cancer were highly heterogeneous. Both overlapping and distinct populations of low abundant fibroblast subtypes in benign prostate stroma were enriched in Gleason grade 4 prostate cancer with cribriform morphology compared to Gleason grade 4 prostate cancer with noncribriform morphology and Gleason grade 3 prostate cancer. In addition, gene expression was distinctly altered in CAF subtypes adjacent to cribriform prostate cancer. Overall, these studies suggest that cribriform prostate cancer has a unique tumor microenvironment that may distinguish it from other Gleason grade 4 morphologies and lower Gleason grades.

Keywords: ASPN; ENG; FAP; NT5E; PDGFRβ; RNAscope; THY1; cancer; cancer-associated fibroblasts; cribriform; prostate; stroma; tumor microenvironment.

Figure 1

ASPN and FAP expression in cribriform prostate cancer. (A) Representative images of dual ASPN (blue) and FAP (red) mRNA expression in stroma adjacent to benign prostate, Gleason grade 3 prostate cancer, Gleason grade 4 prostate cancer without cribriform morphology, and Gleason grade 4 prostate cancer with cribriform morphology (×400 magnification). (B) The percentage of ASPN ⁺ stromal cells adjacent to benign prostate (n = 21), Gleason grade 3 prostate cancer (n = 13), Gleason grade 4 prostate cancer without cribriform (n = 17), and Gleason grade 4 prostate cancer with cribriform morphology (n = 12). (C) The relative expression of ASPN in ASPN ⁺ stromal cells. (D) The mean ASPN H‐score. (E) The percentage of FAP ⁺ stromal cells adjacent to benign prostate (n = 11), Gleason grade 3 prostate cancer (n = 5), Gleason grade 4 prostate cancer without cribriform morphology (n = 8), and Gleason grade 4 prostate cancer with cribriform morphology (n = 6). (F) The relative expression of FAP in FAP ⁺ stromal cells. (G) The mean FAP H‐score. (H) The percent ASPN ⁺ FAP ⁻, ASPN ⁻ FAP ⁺, and ASPN ⁺ FAP ⁺ stromal cells adjacent to benign prostate (n = 11), Gleason grade 3 prostate cancer (n = 5), Gleason 4 prostate cancer without cribriform morphology (n = 8), and Gleason grade 4 prostate cancer with cribriform morphology (n = 6). Gene expression detected by RNAScope® and quantified with Halo® Software. Statistical analyses performed using one‐way analysis of variance with Tukey multiple comparisons. Graphs shown as mean ± SEM, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001.

Figure 2

ASPN and THY1 expression in cribriform prostate cancer. (A) Representative images of dual ASPN (blue) and THY1 (red) mRNA expression in stroma adjacent to benign prostate (n = 10), Gleason grade 3 prostate cancer (n = 6), Gleason grade 4 prostate cancer without cribriform morphology (n = 6), and Gleason grade 4 prostate cancer with cribriform morphology (n = 7) (×400 magnification). (B) The percentage of THY1 ⁺ stromal cells adjacent to benign prostate, Gleason grade 3 prostate cancer, Gleason grade 4 prostate cancer without cribriform morphology, and Gleason grade 4 prostate cancer with cribriform morphology. (C) The relative expression of THY1 in THY1 ⁺ stromal cells. (D) The mean THY1 H‐score. (E) The percent ASPN ⁺ THY1 ⁻, ASPN ⁻ THY1 ⁺, and ASPN ⁺ THY1 ⁺ stromal cells adjacent to benign prostate, Gleason grade 3 prostate cancer, Gleason grade 4 prostate cancer without cribriform morphology, and Gleason grade 4 prostate cancer with cribriform morphology. Gene expression detected by RNAScope® and quantified with Halo® Software. Statistical analyses performed using one‐way analysis of variance with Tukey multiple comparisons. Graphs shown as mean ± SEM, *p ≤ 0.05, ****p < 0.0001.

Figure 3

ASPN and ENG expression in cribriform prostate cancer. (A) Representative images of dual ASPN (red) and ENG (blue) mRNA expression in stroma adjacent to benign prostate (n = 10), Gleason grade 3 prostate cancer (n = 6), Gleason grade 4 prostate cancer without cribriform morphology (n = 7), and Gleason grade 4 prostate cancer with cribriform morphology (n = 6) (×400 magnification). (B) The percentage of ENG ⁺ stromal cells adjacent to benign prostate, Gleason grade 3 prostate cancer, Gleason grade 4 prostate cancer without cribriform morphology, and Gleason grade 4 prostate cancer with cribriform morphology. (C) The relative expression of ENG in ENG ⁺ stromal cells. (D) The mean ENG H‐score. (E) The percent ASPN ⁺ ENG ⁻, ASPN ⁻ ENG ⁺, and ASPN ⁺ ENG ⁺ stromal cells adjacent to benign prostate, Gleason grade 3 prostate cancer, Gleason grade 4 prostate cancer without cribriform morphology, and Gleason grade 4 prostate cancer with cribriform morphology. Gene expression detected by RNAScope® and quantified with Halo® Software. Statistical analyses performed using one‐way analysis of variance with Tukey multiple comparisons. Graphs shown as mean ± SEM, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001.

Figure 4

ASPN and NT5E expression in cribriform prostate cancer. (A) Representative images of dual ASPN (blue) and NT5E (red) mRNA expression in stroma adjacent to benign prostate (n = 9), Gleason grade 3 prostate cancer (n = 6), Gleason grade 4 prostate cancer without cribriform morphology (n = 6), and Gleason grade 4 prostate cancer with cribriform morphology (n = 6) (×400 magnification). (B) The percentage of NT5E ⁺ stromal cells adjacent to benign prostate, Gleason grade 3 prostate cancer, Gleason grade 4 prostate cancer without cribriform morphology, and Gleason grade 4 prostate cancer with cribriform morphology. (C) The relative expression of NT5E in NT5E ⁺ stromal cells. (D) The mean NT5E H‐score. (E) The percent ASPN ⁺ NT5E ⁻, ASPN ⁻ NT5E ⁺, and ASPN ⁺ NT5E ⁺ stromal cells adjacent to benign prostate, Gleason grade 3 prostate cancer, Gleason grade 4 prostate cancer without cribriform morphology, and Gleason grade 4 prostate cancer with cribriform morphology. Gene expression detected by RNAScope® and quantified with Halo® Software. Statistical analyses performed using one‐way analysis of variance with Tukey multiple comparisons. Graphs shown as mean ± SEM, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 5

ASPN and TNC expression in cribriform prostate cancer. (A) Representative images of dual ASPN (red) and TNC (blue) mRNA expression in stroma adjacent to benign prostate (n = 7), Gleason grade 3 prostate cancer (n = 4), Gleason grade 4 prostate cancer without cribriform morphology (n = 6), and Gleason grade 4 prostate cancer with cribriform morphology (n = 5) (×400 magnification). (B) The percentage of TNC ⁺ stromal cells adjacent to benign prostate, Gleason grade 3 prostate cancer, Gleason grade 4 prostate cancer without cribriform morphology, and Gleason grade 4 prostate cancer with cribriform morphology. (C) The relative expression of TNC in TNC ⁺ stromal cells. (D) The mean TNC H‐score. (E) The percent ASPN ⁺ TNC ⁻, ASPN ⁻ TNC ⁺, and ASPN ⁺ TNC ⁺ stromal cells adjacent to benign prostate, Gleason 3 prostate cancer, Gleason 4 without cribriform morphology prostate cancer, and Gleason 4 with cribriform morphology prostate cancer. Gene expression detected by RNAScope® and quantified with Halo® Software. Statistical analyses performed using one‐way analysis of variance with Tukey multiple comparisons. Graphs shown as mean ± SEM, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 6

ASPN and PDGFRβ expression in cribriform prostate cancer. (A) Representative images of dual ASPN (blue) and PDGFRβ (red) mRNA expression in stroma adjacent to benign prostate (n = 9), Gleason grade 3 prostate cancer (n = 6), Gleason grade 4 prostate cancer without cribriform morphology (n = 6), and Gleason grade 4 prostate cancer with cribriform morphology (n = 7) (×400 magnification). (B) The percentage of PDGFRβ ⁺ stromal cells adjacent to benign prostate, Gleason grade 3 prostate cancer, Gleason grade 4 prostate cancer without cribriform morphology, and Gleason grade 4 prostate cancer with cribriform morphology. (C) The relative expression of PDGFRβ in PDGFRβ ⁺ stromal cells. (D) The mean PDGFRβ H‐score. (E) The percent ASPN ⁺ PDGFRβ ^‐, ASPN ⁻ PDGFRβ ⁺, and ASPN ⁺ PDGFRβ ⁺ stromal cells adjacent to benign prostate, Gleason grade 3 prostate cancer, Gleason grade 4 prostate cancer without cribriform morphology, and Gleason grade 4 prostate cancer with cribriform morphology. Gene expression detected by RNAScope® and quantified with Halo® Software. Statistical analyses performed using one‐way analysis of variance with Tukey multiple comparisons. Graphs shown as mean ± SEM, *p ≤ 0.05, ***p ≤ 0.001, ****p < 0.0001.