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. 2017 Aug 14;32(2):253-267.e5.
doi: 10.1016/j.ccell.2017.07.006.

Macrophage Polarization Contributes to Glioblastoma Eradication by Combination Immunovirotherapy and Immune Checkpoint Blockade

Dipongkor Saha  1 , Robert L Martuza  1 , Samuel D Rabkin  2
Affiliations

Macrophage Polarization Contributes to Glioblastoma Eradication by Combination Immunovirotherapy and Immune Checkpoint Blockade

Dipongkor Saha et al. Cancer Cell. .

Abstract

Glioblastoma is an immunosuppressive, fatal brain cancer that contains glioblastoma stem-like cells (GSCs). Oncolytic herpes simplex virus (oHSV) selectively replicates in cancer cells while inducing anti-tumor immunity. oHSV G47Δ expressing murine IL-12 (G47Δ-mIL12), antibodies to immune checkpoints (CTLA-4, PD-1, PD-L1), or dual combinations modestly extended survival of a mouse glioma model. However, the triple combination of anti-CTLA-4, anti-PD-1, and G47Δ-mIL12 cured most mice in two glioma models. This treatment was associated with macrophage influx and M1-like polarization, along with increased T effector to T regulatory cell ratios. Immune cell depletion studies demonstrated that CD4+ and CD8+ T cells as well as macrophages are required for synergistic curative activity. This combination should be translatable to the clinic and other immunosuppressive cancers.

Keywords: HSV; cancer stem cells; glioma; immunotherapy; oncolytic virus.

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Figures

Figure 1
Figure 1. Characterization of mouse 005 and human GSCs
A. 005 GSCs cultured for 24 hr with or without murine IFNγ (mIFNγ: 0 or 3 ng/ml), stained for PD-L1, and analyzed by flow cytometry. B. 005 GSCs infected with G47Δ-E or G47Δ-mIL12 at MOI=1 for 24 hr, stained for PD-L1, and analyzed by flow cytometry. C. Human primary (left) and recurrent (right) GSCs cultured for 24 hr, stained for PD-L1 (cyan), and analyzed by flow cytometry. Percent of PD-L1+ cells indicated in upper right. See also Figure S1.
Figure 2
Figure 2. Immunohistochemical staining of immune cell markers after G47
(A–B) Δ-mIL12 treatment. (A–B) 005 GSCs (2 × 104) implanted on day 0, injected intratumorally (IT) with G47Δ-mIL12 (1 × 105 pfu; n=3) or PBS (n=4) on days 18 and 24, animals sacrificed on day 25, and brains collected. Brain tumor sections were stained as indicated. For PD-L1 brain sections, 005 GSCs (2 × 104) implanted on day 0, injected IT with G47Δ-mIL12 (5 × 105 pfu; n=3) or PBS (n=3) on days 17, animals sacrificed on day 24, and brain tumor sections stained with anti-PD-L1 antibody. Representative images are presented; scale bar=100 μm (A). The number of positive cells from 3–9 fields/tumor section (1 section/mouse, except 3 for CD3) were counted (B). Average number of positive cells from each individual mouse is identified by symbol and color. The mean ± SEM of all mice is presented. Data were assessed by Student’s t test; *p<0.05; **p<0.001. See also Figure S1.
Figure 3
Figure 3. Treatment of 005 GSC-derived tumors with systemic immune checkpoint inhibitors, intratumoral G47 Δ-mIL12, or the combination prolongs survival
(A–B) Mice implanted with 2 × 104 005 GSCs on day 0, treated with G47Δ-mIL12 (5 × 105 pfu) or PBS injected IT on day 12 (upward arrow) and isotype control IgG (10 mg/kg), anti-(α)PD-L1 antibody (A), or anti-(α)PD-1 antibody (B) injected IP on days 15, 18 and 21 (downward arrows). Values from a single experiment, with Mock (treated with PBS and IgG) and G47Δ-mIL12 groups the same in A and B. Median survival of Mock (33.5 days; n=6) was compared to anti-PD-1 (39 days; n=8, p=0.02), anti-PD-L1 (42 days; n=7, p=0.003), or G47Δ-mIL12 (39 days; n=8, p=0.01) by Log-rank analysis. Similarly, G47Δ-mIL12 was compared to the combination of G47Δ-mIL12 with anti-PD-1 (49 days; n=7, p=0.02) or -PD-L1 (50 days; n=7, p=0.03), and antibodies were compared to the combination of G47Δ-mIL12 with anti-PD-1 (p=0.053) or anti-PD-L1 (p=0.08). Experiment was conducted once (A) or twice (B). C. Mice implanted with 2 × 104 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-(α)CTLA-4 antibody or isotype control IgG (5 mg/kg) injected IP on days 8, 11 and 14 (n=8/group, except for G47Δ-mIL12 n=7). Median survival of Mock (37.5 days) was compared to anti-CTLA-4 (45 days; p=0.002) or G47Δ-mIL12 (40 days; p=0.03) alone by Log-rank analysis. Similarly, combination of G47Δ-mIL12 with anti-CTLA-4 (58 days) was compared to anti-CTLA-4 (p=0.05) or G47Δ-mIL12 (p=0.008). Experiment was conducted 2 times. D. Mice implanted with 2 × 104 005 GSCs on day 0, treated with PBS (right; n=2), rat anti-(α)PD-1 antibody (middle; 200 μg/mouse; n=2), or rat anti-(α)PD-L1 antibody (left; 200 μg/mouse; n=2) injected IP on day 25, and sacrificed 3 hr later. Antibodies were detected with HRP-conjugated anti-(α)rat Ig (brown; right) or control HRP-conjugated anti-(α)rabbit Ig (left). * normal brain adjacent to tumor. Scale bar=100 μm. E. 005 GSCs (1.5 × 105) implanted on day 0, treated with PBS or G47Δ-mIL12 (5 × 105 pfu) IT on day 11 and IgG or anti-CTLA-4 antibody (5 mg/kg) injected IP on days 11, 14, and 17 (n=4/group; Mock=PBS/IgG), and mice sacrificed on day 18. Tumors were harvested, cells stained with fluorochrome-conjugated anti-mouse antibodies, and multicolor FACS performed. Scatter plot (each animal 1 point) of the percentages of live sorted positive cells. The ratio of Teff to Treg ratio is presented in a bar graph. Mean ± SEM. Data were assessed by Student’s t test between indicated groups; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Figure S2 and S3.
Figure 4
Figure 4. Triple combination therapy in 005 and CT-2A glioma models
A. Mice implanted with 2 × 104 005 GSCs on day 0 and treated with G47Δ-mIL12 (5 × 105 pfu) or PBS injected IT on day 8 (upward arrow) and anti-CTLA-4 (5 mg/kg) and anti-PD-1 (10 mg/kg) or isotype control IgG (5 mg/kg hamster and 10 mg/kg rat IgG) IP on days 8, 11 and 14 (n=9/group; downward arrows). Median survival of Mock (PBS and IgG; 40 days) was compared to the combination of anti-PD-1 and anti-CTLA-4 (55 days; p=0.0002) by Log-rank analysis. Similarly, triple combination treatment with virus (89% of mice surviving long-term) was compared to the combination of anti-PD-1 and anti-CTLA-4 (p<0.0001) or Mock (p<0.0001). Experiment was conducted 3 times. B. Cured mice (n=8) from the triple combination experiment in (A) were re-challenged on day 183 with 5-fold increased number of 005 GSCs (1 × 105) in the contralateral hemisphere (re-challenged). Age matched (8 months) naive mice were implanted as controls (n=5). All re-challenged mice were alive at day 96 post-challenge without tumor; controls had a median survival of 27 days post-challenge (p=0.0001; Log-rank analysis). C. CT-2A cells (1 × 104) implanted in C57Bl/6 mice on day 0, injected IT with G47Δ-mIL12 (1 × 105 pfu) or PBS on day 10 and anti-CTLA-4 antibody and anti-PD-1 antibody or isotype control IgG (5 mg/kg hamster IgG and 10 mg/kg rat IgG) injected IP on days 10, 13 and 16. Median survival of Mock (PBS/IgG) treated mice (20 days; n=9) was compared to G47Δ-mIL12 (21 days; n=8) by Log-rank analysis (p=0.007). Median survival of mice treated with triple combination (66.5 days; 50% of mice surviving long-term) was compared to anti-PD-1 and -CTLA-4 (19 days; n=8, p=0.005) or G47Δ-mIL12 (p=0.01). D. Cured mice (n=4) from the triple combination experiment in (C) were re-challenged on day 109 with 5-fold increased number of CT-2A cells (5 × 104). Similar age naive mice (~6 months) were implanted as controls (n=5). All re-challenged mice were alive at day 46 post-challenge without tumor, and compared to control by Log-rank analysis (p=0.005). See also Figure S4.
Figure 5
Figure 5. FACS analysis of treatment effects of triple combination therapy
(A–C) Mice implanted with 005 GSCs (1.5 × 105) on day 0, treated with G47Δ-mIL12 (5 × 105 pfu) or PBS injected IT on day 11, and anti-PD-1 and anti-CTLA-4 antibody or rat and hamster IgGs (Mock) (5 mg/kg hamster IgG and 10 mg/kg rat IgG) injected IP on days 11, 14, and 17 (n=4), and sacrificed on day 18. Tumors harvested, dissociated cells stained with fluorochrome-conjugated anti-mouse antibodies, and multicolor FACS was performed. Percentages of live CD3+ cells, CD3+ sorted CD4+ and CD8+ subsets, live CD11b+ (monocytes, macrophages, NK, DC), and live CD45− GFP+ 005 cells from 2 independent experiments (red and black symbols) for M, CPi, V, and CPi+V groups (symbols are individual mice) were analyzed (A). The tumors from the experiment with red symbols in (A) were analyzed for CD3+FoxP3+ subtypes and ratio of CD8+ to CD4+FoxP3+ (B). The tumors from the experiment with black symbols in (A) were analyzed for immune checkpoint expression (PD-L1, CTLA-4, PD-1) on CD45+CD3+CD4+ and CD8+ subsets, CD45−GFP+, and CD11b+CD45hi and lo cells (C). M, Mock (PBS/IgG); V, virus (G47Δ-mIL12); CPi, checkpoint inhibitors (anti-PD-1+anti-CTLA-4); CPi+V, virus + checkpoint inhibitors (G47Δ-mIL12+anti-PD-1+anti-CTLA-4). Data are mean ± SEM and assessed by Student’s t test between indicated groups; *p<0.05, **p<0.01, ***p<0.001. See also Figure S5.
Figure 6
Figure 6. Immunohistochemical staining of tumor infiltrating cells
(A–D) Mice implanted with 005 GSCs (2 × 104) on day 0, treated with G47Δ-mIL12 (5 × 105 pfu) or PBS injected IT on day 17, and checkpoint inhibitors anti-PD-1 antibody and anti-CTLA-4 antibody or rat and hamster IgGs (5 mg/kg hamster IgG and 10 mg/kg rat IgG) injected IP on days 17, 20, and 23 (n=4), sacrificed on day 24, and brain tumor sections stained as indicated (Mock; PBS/IgG). Representative images with positive cells stained brown are presented (A). PD-L1 staining was from a separate experiment, but same treatment as above (n=3 or 4). Scale bars=100 or 200 μm as indicated. Number of positive cells per field in (A) were counted (3–5 fields/section/mouse for CD68 and Ki67; 8–10 fields/section/mouse for pStat1 and iNOS; 4 fields/section and 2 sections/mouse for PD-L1), and individual mice in each group are identified by color (B). Representative images with positive cells stained red (pSTAT1 +, CD3+), blue (CD68+, Ki67+), and red/blue colocalized (examples indicated with arrows) are presented (C). Scale bar=100 μm. Brain sections were incubated sequentially with primary (pSTAT1 or CD3 rabbit antibody) and secondary antibodies (AP-conjugated anti-rabbit Ig), followed by red color development. The same sections were then incubated with primary (CD68 or Ki67; rabbit antibody) and secondary antibodies (AP-conjugated anti-rabbit Ig), followed by blue color development. Number of positive cells per field were counted (8–10 fields/section/mouse) and percent of double positive cells plotted (D) with individual mice identified by color. M, Mock (PBS/IgG); CPi, checkpoint inhibitors (anti-PD-1+anti-CTLA-4); CPi+V, checkpoint inhibitors + virus (G47Δ-mIL12+anti-PD-1+anti-CTLA-4). Data are mean ± SEM, assessed by Student’s t test between indicated groups; *p<0.05, ***p<0.001, ****p<0.0001. See also Figure S6.
Figure 7
Figure 7. Depletion/inhibition of immune cell subtypes abrogates triple combination therapy
A. C57Bl/6 mice implanted with (2 × 104) 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-CTLA-4 and anti-PD-1 antibodies or isotype control IgG (5 mg/kg hamster IgG and 10 mg/kg rat IgG) injected IP on days 8, 11 and 14 (n=6/group; upward arrows). Depletion antibodies against CD4 or CD8 (10 mg/kg) or clodronate liposomes (Clod; first injection 50 mg/kg followed by 25 mg/kg) were injected IP on days 4, 7, 10, 13, 20, and 27 (downward arrows), or BLZ945 (BLZ; 200 mg/kg) was gavaged for two cycles from days 6–10 and days 12–16 in triple therapy mice. Median survival of mice was determined: Mock (PBS/IgG/liposome/20% captisol), 35.5 days or triple therapy +αCD4 antibody, 32.5 days; +BLZ, 41.5 days; +αCD8 antibody, 45 days; +Clod, 43 days; +PBS (IgG/liposome/20% captisol). +PBS was compared to +BLZ (p=0.004), +Clod (p=0.02), +αCD4 (p=0.0007), +αCD8 (p=0.02), or Mock (p=0.0006) by Log-rank analysis. Similarly, Mock was compared to +PBS (p=0.0006), +BLZ (p=0.06), +Clod (p=0.01), +αCD4 (p=0.4), or +αCD8 (p=0.0006). B. C57Bl/6 mice implanted with 005 GSCs (2 × 104) on day 0 and treated with G47Δ-mIL12 (5 × 105 pfu) or PBS injected IT on day 18 and anti-CTLA-4 and anti-PD-1 antibodies or isotype control IgGs (5 mg/kg hamster IgG and 10 mg/kg rat IgG) injected IP on days 18, 21 and 24 (n=2/group). Depletion antibodies (αCD4, αCD8; 10 mg/kg) or Clod (first injection 50 mg/kg followed by 25 mg/kg) were injected IP on days 14, 17, 20, and 23, or BLZ (200 mg/kg) gavaged from days 16–20 and 22–25. Twenty-four hr after the last immune checkpoint injection or 8 hr after the last BLZ gavage, animals were sacrificed on day 25 and brains collected. Brain tumor sections (2 sections/mouse, at least 200 μm apart from each other) were stained for CD4, CD8, CD68, and F4/80. Positive cells were counted (5 fields/section for CD4+ and CD8+, 8 fields/section for CD68+, and 6 fields/section for F4/80+) and presented as mean ± SEM. Data were assessed by Student’s t test between indicated groups *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Only significant differences between Mock or triple therapy and other treatments indicated. See also Figure S7.
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