Intratumoral mediated immunosuppression is prognostic in genetically engineered murine models of glioma and correlates to immunotherapeutic responses

Abstract

Purpose: Preclinical murine model systems used for the assessment of therapeutics have not been predictive of human clinical responses, primarily because their clonotypic nature does not recapitulate the heterogeneous biology and immunosuppressive mechanisms of humans. Relevant model systems with mice that are immunologically competent are needed to evaluate the efficacy of therapeutic agents, especially immunotherapeutics.

Experimental design: Using the RCAS/Ntv-a system, mice were engineered to coexpress platelet-derived growth factor B (PDGF-B) receptor + B-cell lymphoma 2 (Bcl-2) under the control of the glioneuronal specific Nestin promoter. The degree and type of tumor-mediated immunosuppression were determined in these endogenously arising gliomas on the basis of the presence of macrophages and regulatory T cells. The immunotherapeutic agent WP1066 was tested in vivo to assess therapeutic efficacy and immunomodulation.

Results: Ntv-a mice were injected with RCAS vectors to express PDGF-B + Bcl-2, resulting in both low- and high-grade gliomas. Consistent with observations in human high-grade gliomas, mice with high-grade gliomas also developed a marked intratumoral influx of macrophages that was influenced by tumor signal transducer and activator of transduction 3 (STAT3) expression. The presence of intratumoral F4/80 macrophages was a negative prognosticator for long-term survival. In mice coexpressing PDGF-B + Bcl-2that were treated with WP1066, there was 55.5% increase in median survival time (P < 0.01), with an associated inhibition of intratumoral STAT3 and macrophages.

Conclusions: Although randomization is necessary for including mice in a therapeutic trial, these murine model systems are more suitable for testing therapeutics, especially immunotherapeutics, in the context of translational studies.

Fig. 1

(A) Representative hematoxylin and eosin stained light microscopy images showing the phenotypic features of de novo (i) low- (50X) and (ii) high-grade gliomas (50X) in the PDGF-B + Bcl-2 mouse, including (i, ii) diffuse infiltration, (ii) pseudopallisading necrosis (arrow demonstrating), (iii) white matter tracking (100X), and (iv) vascular proliferation (400X). (B) The induced tumors in the PDGF-B + Bcl-2 mice demonstrating positive expression of (i) GFAP, the (ii) the hemagluttinin epitope tag (detecting PDGF-B expression), and (iii) Bcl-2 (400X; scale bar = 100 µm). (C) Kaplan-Meier survival curves showing overall survival of PDGF-B alone, and PDGF-B + Bcl-2 mice. Median survival was longer than 90 days in the PDGF-B alone mice and 52.5 days in the PDGF-B + Bcl-2 mice (P = 0.005) (Animals surviving for longer than 90 days were terminated for histopathological tissue examination and analysis.)

Fig. 2

(A) Representative light microscopy images showing immunohistochemical staining for p-STAT3 in a high-grade glioma at (i) low (100X) and (ii) high (400X) magnification, and in a low-grade tumor at (iii) low (100X) and (iv) high (400X) magnification, and (v) rabbit IgG isotype control (100X). (B) In the PDGF-B alone, and PDGF-B + Bcl-2 mice that developed gliomas, p-STAT3 levels were significantly higher in high-grade tumors than in low-grade tumors (* P < 0.05). (C) In PDGF-B + Bcl-2 mice with high-grade gliomas, animals that survived past 90 days had significantly lower tumor p-STAT3 expression than animals that succumbed to intracranial tumors before 90 days (** P < 0.0001). (D) Mean p-STAT3 expression in individual mice in the PDGF-B + Bcl2 group bearing high-grade tumors plotted against overall survival time showing long-term survival of all animals with p-STAT3 expression that was < 10%. (* = animals sacrificed after surviving past 90 days. Error bars represent standard error of the mean.)

Fig. 3

(A) Representative light microscopy images showing immunohistochemical staining of F4/80 to identify infiltrating macrophages/microglia in a high-grade tumor at (i) low (100X) and (ii) high (400X) magnification, and a low-grade tumor at (iii) low (100X) and (iv) high (400X) magnification, and (v) rat IgG isotype control (100X). (B) In the PDGF-B alone, and PDGF-B + Bcl-2 mice, macrophage infiltration was significantly higher in high-grade tumors than in low-grade tumors (** P < 0.05). (C) In PDGF-B + Bcl-2 mice with high-grade gliomas that survived past 90 days had significantly lower levels of macrophage infiltration than those that succumbed to intracranial tumors before 90 days (** P = 0.003). (D) Mean degree of macrophage infiltration in individual mice in the PDGF-B + Bcl2 group bearing high-grade tumors plotted against overall survival time, showing long-term survival of all animals with macrophage infiltration of < 20%. (* = animals sacrificed after surviving past 90 days. Error bars represent standard error of the mean.)

Fig. 4

(A) Representative light microscopy images showing immunohistochemical staining of FoxP3 to identify infiltrating Tregs in a high-grade tumor at (i) low (100X) and (ii) high (400X) magnification, and a low-grade tumor at (iii) low (100X) and (iv) high (400X) magnification, and (v) mouse IgG isotype control (100X). (B) In the PDGF-B alone, and PDGF-B + Bcl-2 mice, the level of Treg infiltration was observed to be similar in high- and low-grade tumors. (C) In the PDGF-B + Bcl2 group, no significant differences were observed in mean levels of Treg infiltration between mice with high-grade gliomas who survived longer than 90 days and those that succumbed to intracranial tumors before 90 days. (D) In the PDGF-B + Bcl2 group, the mean degree of Treg infiltration in individual high-grade tumor bearing mice did not correlate with length of survival.

Fig. 5

(A) Kaplan-Meier survival curve showing improved survival in PDGF-B and Bcl-2 mice treated with the p-STAT3 inhibitor WP1066 compared with untreated controls. (B) WP1066 treatment significantly reduced levels of p-STAT3 expression. Representative light microscopy images show immunohistochemistry of p-STAT3 (brown nuclear staining) in a high-grade glioma from a vehicle control mouse and a WP1066-treated mouse (400X). (C) WP1066 also suppressed F4/80-positive macrophage infiltration. Representative light microscopy images showing immunohistochemistry of F4/80 (brown cytoplasmic staining) demonstrating infiltrating macrophages/microglia in a high-grade glioma from a vehicle control mouse and a WP1066-treated mouse (400X). (D) WP1066 did not suppress the number of intratumoral FoxP3-positive cells. Representative light microscopy images showing immunohistochemistry of FoxP3 (brown staining) from a vehicle control mouse and a WP1066-treated mouse (400X). (* P < 0.05).