Tumor-targeted TNFα stabilizes tumor vessels and enhances active immunotherapy - PubMed (original) (raw)

Tumor-targeted TNFα stabilizes tumor vessels and enhances active immunotherapy

Anna Johansson et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Solid tumors are intrinsically resistant to immune rejection. Abnormal tumor vasculature can act as a barrier for immune cell migration into tumors. We tested whether targeting IFNγ and/or TNFα into pancreatic neuroendocrine tumors can alleviate immune suppression. We found that intratumoral IFNγ causes rapid vessel loss, which does not support anti-tumor immunity. In contrast, low-dose TNFα enhances T-cell infiltration and overall survival, an effect that is exclusively mediated by CD8(+) effector cells. Intriguingly, lymphocyte influx does not correlate with increased vessel leakiness. Instead, low-dose TNFα stabilizes the vascular network and improves vessel perfusion. Inflammatory vessel remodeling is, at least in part, mediated by tumor-resident macrophages that are reprogrammed to secrete immune and angiogenic modulators. Moreover, inflammatory vessel remodeling with low-dose TNFα substantially improves antitumor vaccination or adoptive T-cell therapy. Thus, low-dose TNFα promotes both vessel remodeling and antitumor immune responses and acts as a potent adjuvant for active immunotherapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

IFNγ and TNFα have distinct effects in the tumor microenvironment. (A) Schematic representation of a short-term treatment regimen in RIP1-Tag5 mice. Arrows indicate four i.v. injections of compounds. Tumors were analyzed at 29 wk. (B) Costaining of control (untreated), IFNγ–RGR and TNFα-RGR treated tumors with specific antibodies: CD8+ T cells, red; CD31+ blood vessels, green. Representative pictures after biweekly i.v. injections of 2 μg of IFNγ–RGR or TNFα-RGR for 2 wk are shown. (Original magnification: 20×) (Scale bar: 100 μm.) (C) Quantification of tumor-infiltrating CD8+ T cells (mean CD8+ T cells per field ± SE, n = 3–9, *P < 0.01 compared with all other groups). (D) Quantification of CD31-positive blood vessels (mean % of CD31-covered area/field ± SE, n = 4–6, *P ≤ 0.01 compared with all other treatment groups). (E) Costaining of CD31-positive blood vessels (red) with TUNEL+, apoptotic cells (green) in IFNγ–RGR treated tumors. (Original magnification: 10×) (Scale bar: 200 μm.) Inset shows clustering of apoptotic cells around a vessel. (Original magnification: 40×) (Scale bar: 50 μm.) (F) Quantification of apoptotic cells in different treatment groups (mean TUNEL+ cells per field ± SE, n = 3–7, *P = 0.02 compared with control and TNFα-RGR treated groups).

Fig. 2.

Fig. 2.

Long-term survival under TNFα-RGR monotherapy is CD8+ T-cell dependent. (A) Untreated or TNFα/TNFα-RGR treated RIP1-Tag5/F1 mice were assessed for in vivo CTL activity against the Tag-specific peptide IV after 2 wk of treatment. (Left) Combined data for spleen cells (n = 5) and tumor-draining pancreatic lymph nodes (LN; n = 3–5, LNs were pooled in each of two independent experiments). (Right) Representative histograms of percent specific kill of CFSEhigh LN cells from two treatment groups. (B) Long-term treatment scheme: RIP1-Tag5 mice were treated at the age of 22–23 wk with biweekly i.v. injections and survival monitored. Percent survival of RIP1-Tag5 mice treated with 2 μg of TNFα or TNFα-RGR (P = 0.002, TNFα-RGR compared with TNFα; P = 0.001, TNFα-RGR compared with untreated controls (n = 5–7) (C), and 2 μg of TNFα-RGR in the presence (αCD8) and absence (IgG) of CD8+ T-cell depleting antibodies (P = 0.0002, TNFα-RGR plus depletion compared with TNFα-RGR with control IgG, n = 8) (D).

Fig. 3.

Fig. 3.

Intratumoral TNFα-RGR enhances efficacy of anticancer immunotherapy. (A) RIP1-Tag5 mice were treated with vaccine alone, 2 μg of TNFα-RGR alone or in combination with anti-Tag vaccine (P = 0.007 single versus combination treatment, n = 8–12). (B) RIP1-Tag5 mice were treated every second week with adoptive transfers (ad T) of preactivated CD4+ and CD8+ Tag-specific T cells alone or in combination with 2 μg of TNFα-RGR, survival was monitored up to 45 wk (P < 0.0001, n = 8–10). (C) Percentage of TagTCR8 T cells in pancreatic lymph nodes (panc LN) or tumors was tracked by FACS analysis for 21 d after adoptive transfer in untreated (Left) or TNFα-RGR treated mice (Right), n = 3.

Fig. 4.

Fig. 4.

Tumor-targeted TNFα stabilizes vessels and enhances vascular functionality. (A) Representative pictures of CD31-positive vessels in control (untreated) RIP1-Tag5 tumors and after 2 wk of treatment with 2 μg of TNFα-RGR. Arrows point at large vessels. (Original magnification: 20×.) (Scale bar: 100 μm.) (B) Quantification of mean vessel length in control (Ctrl) and treatment groups (T-R, TNFα-RGR) (P = 0.01). (C) Quantification of percentage of large vessels (size: 150–200 μm) (P = 0.003). (D Upper) CD31+ vessels (green) and coverage with PDGFRβ+ pericytes (red). Arrow points at a pericyte-covered area in controls. (Lower) CD31+ vessels (red) and association of αSMA+ perivascular cells (green). Arrow points at close vascular lining in TNFα-RGR treated tumors. (Original magnification: 40×.) (Scale bar: 50 μm.) (E) Ratio of PDGFRβ-positive pericytes to CD31-positive endothelial cells (P = 0.003). (F) Percent αSMA+ covered endothelial cells (P = 0.02). (G) Vascular permeability assessed by injection of 70-kDa Texas-red labeled dextran followed by saline perfusion. (Upper) Dextran signals in tumors. Arrows point at areas of dextran extravasation. Arrowheads point at residual dextran associated with vessels. (Lower) Dextran/dapi double staining. (Original magnification: 20×.) (Scale bar: 100 μm.) (H) Quantification of percentage of dextran in tumors as readout for vascular leakiness (P = 0.02). (I) CD31-positive vessels (Upper) in relation to i.v. injected FITC-lectin (Lower). Dashed line indicates perfused and nonperfused tumor areas. (Original magnification: 20×.) (Scale bar: 100 μm.) (J) Ratio lectin-positive vessels to CD31-positive vessels (P = 0.03, n = 3–8 for all groups).

Fig. 5.

Fig. 5.

Tumor macrophages are activated and reprogrammed to express immunostimulatory factors and angiogenic modulators. (A) Quantitative PCR analysis of isolated CD68+ macrophages from TNFα-RGR treated tumors, expressed as fold change relative to CD68+ from control (untreated) RIP1-Tag5 tumors (n = 3). (B Left) Quantitative analysis of TagTCR8 cell proliferation, unstimulated (-) or stimulated with Tag-specific peptide/IL2 (+) in the presence of macrophages (MØ) isolated from untreated controls (ctrl) or tumors after 2 wk of treatment with 2 μg of TNFα-RGR (T-R) (P = 0.01). (Right) Representative histograms showing percent proliferation of CFSE-labeled T cells from all groups. (C Left) HUVEC were incubated with macrophages isolated from untreated (MØ ctrl) or TNFα-RGR treated tumors (MØ T-R) in the presence of Ang2 receptor (Tie2) (Ang2 block) or TNFα blocking antibodies (TNFα block). Arrows delineate VCAM positive, cellular HUVEC staining. (Original magnification: 40×.) (Scale bar: 50 μm.) (C Right) Quantification of percent VCAM-positive cells in relation to DAPI-positive cells (P = 0.08).

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