Presence of cancer-associated fibroblasts inversely correlates with Schwannian stroma in neuroblastoma tumors

Abstract

Stromal cells have a central function in the regulation of tumor angiogenesis. Recent studies have shown that stromal myofibroblasts (cancer-associated fibroblasts) actively promote tumor growth and enhance tumor angiogenesis in many types of adult carcinomas. To evaluate the function cancer-associated fibroblasts have in neuroblastoma angiogenesis and investigate their relationship to stromal Schwann cells, we quantified cancer-associated fibroblasts in 60 primary neuroblastoma tumors and in a novel neuroblastoma xenograft model in which murine Schwann cells were induced to infiltrate into the tumor stroma. Tumor sections were examined for presence of microvascular proliferation, a hallmark of tumor angiogenesis. Cancer-associated fibroblasts were characterized by positive immunostaining for alpha-smooth muscle actin (alpha-SMA) and were distinguished from pericytes by staining negatively for high-molecular-weight caldesmon. alpha-SMA-positive cells were quantified and their number was defined as high when >1.0% of the area was positive. Associations between high cancer-associated fibroblast number, microvascular proliferation and established prognosticators were analyzed. High numbers of cancer-associated fibroblasts were associated with Schwannian stroma-poor histopathology and microvascular proliferation. Thirty-seven (80%) of the 46 Schwannian stroma-poor tumors had high numbers of cancer-associated fibroblasts in the tumor stroma compared to only 2 (14%) of the 14 Schwannian stroma-rich/dominant tumors (P<0.001). Thirty-three (89%) of 37 tumors with microvascular proliferation had high numbers of cancer-associated fibroblasts compared to 9 (40%) of 22 tumors without microvascular proliferation (P<0.001). In the xenografts with infiltrating Schwann cells (n=10), the number of cancer-associated fibroblasts per mm(2) was approximately sevenfold less than in the control xenografts without stromal Schwann cells (n=9) (mean of 51+/-30 vs 368+/-105, respectively; P<0.001). Thus, cancer-associated fibroblasts were inversely associated with presence of Schwann cells, suggesting that Schwann cells may prevent the activation of fibroblasts. A deeper understanding of the function cancer-associated fibroblasts have in neuroblastoma angiogenesis may guide future development of stroma-directed therapeutic strategies.

Fig. 1. Cancer Associated Fibroblasts in Schwannian Stroma-poor neuroblastoma tumors

Representative sections of human ganglioneuroma[GNR] (A-D), ganglioneuroblastoma intermixed[GNBI] (E-H), ganglioneuroblastoma nodular[GNBN] (I-L), and neuroblastoma tumors that are differentiating[NBD] (M-P), poorly differentiated[NBPD] (Q-T), and undifferentiated[NBU] (U-W) tumors stained with H&E (A, E, I, M, Q, and U), Masson Trichrome special stain (B, F, J, N, R, and V), α-SMA which reveals cancer-associated fibroblasts and pericytes (C, G, K, O, S, and W), and high molecular weight caldesmon, which stains pericytes, but not cancer-associated fibroblasts (D, H, L, P, and T). The mean (±standard deviation) percent of α-SMA positive areas per total tumor area analyzed for each tumor type are shown in the bar graph (X). α-SMA positive, caldesmon-negative cancer-associated fibroblasts are rare in the Schwannian Stroma[SS]-dominant ganglioneuroma (C, D and X) and Schwannian Stroma -rich ganglioneuroblastoma intermixed (G/H and X) tumors. Significantly more cancer-associated fibroblasts are present in Schwannian Stroma -poor tumors (K, L, S, T, W). Bands of fibrocollagenous stroma are thin and delicate in ganglioneuroma and ganglioneuroblastoma intermixed tumors, but are thick and support microvascular proliferation in ganglioneuroblastoma nodular and neuroblastoma tumors (pale blue B and F vs. J, N, R and V). Cancer-associated fibroblasts constitute the majority of stromal cells within fibrovascular stroma (O, S and W). Original magnification x200 (A-L, T) and x400 (P).

Fig. 2. Cancer-associated fibroblasts in human neuroblastomas with microvascular proliferation

Representative sections of differentiating neuroblastoma[NBD] (A), poorly differentiated neuroblastoma[NBPD] (B), ganglioneuroma[GNR] (C) and ganglioneuroblastoma intermixed[GNBI] (D) tumors immunostained for α-SMA. The mean (±standard deviation) percent of α-SMA -positive areas per total tumor areas analyzed for tumors with microvascular proliferation[MVP] are shown in the bar graph (E). Significantly more α-SMA -positive cancer-associated fibroblasts are present in the tumors with microvascular proliferation (A, B, and E) as compared to tumors without microvascular proliferation (C, D, and E) (p<0.001). The cancer-associated fibroblasts are present within bands of fibrovascular stroma supporting microvascular proliferation (A and B). Original magnification x400.

Fig. 3. Cancer Associated Fibroblasts are decreased in neuroblastoma xenografts with infiltrating murine Schwann cells

Representative sections of SMS-KCNR tumors engrafted outside (A and B) versus inside (C and D) the sciatic nerve immunostained for α-SMA. The mean (±SD) number of α-SMA -positive cells per mm² of the tumors engrafted outside (n=9) and inside (n=10) the sciatic nerve are shown in the bar graph (E). Significantly more α-SMA -positive Cancer Associated Fibroblasts are present in tumors engrafted outside (A and B) as compared to inside the sciatic nerve (C and D) (p<0.001 for total α-SMA -positive cells). Original magnification x200 (A, C) and x400 (B, D).