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. 2019 Sep 30;93(20):e00533-19.
doi: 10.1128/JVI.00533-19. Print 2019 Oct 15.

Murine Cytomegalovirus Infection of Melanoma Lesions Delays Tumor Growth by Recruiting and Repolarizing Monocytic Phagocytes in the Tumor

Nicole A Wilski  1 , Christina Del Casale  1 , Timothy J Purwin  2 , Andrew E Aplin  2   3 , Christopher M Snyder  4   3
Affiliations

Murine Cytomegalovirus Infection of Melanoma Lesions Delays Tumor Growth by Recruiting and Repolarizing Monocytic Phagocytes in the Tumor

Nicole A Wilski et al. J Virol. .

Abstract

Cytomegalovirus (CMV) is a ubiquitous betaherpesvirus that infects many different cell types. Human CMV (HCMV) has been found in several solid tumors, and it has been hypothesized that it may promote cellular transformation or exacerbate tumor growth. Paradoxically, in some experimental situations, murine CMV (MCMV) infection delays tumor growth. We previously showed that wild-type MCMV delayed the growth of poorly immunogenic B16 melanomas via an undefined mechanism. Here, we show that MCMV delayed the growth of these immunologically "cold" tumors by recruiting and modulating tumor-associated macrophages. Depletion of monocytic phagocytes with clodronate completely prevented MCMV from delaying tumor growth. Mechanistically, our data suggest that MCMV recruits new macrophages to the tumor via the virus-encoded chemokine MCK2, and viruses lacking this chemokine were unable to delay tumor growth. Moreover, MCMV infection of macrophages drove them toward a proinflammatory (M1)-like state. Importantly, adaptive immune responses were also necessary for MCMV to delay tumor growth as the effect was substantially blunted in Rag-deficient animals. However, viral spread was not needed and a spread-defective MCMV strain was equally effective. In most mice, the antitumor effect of MCMV was transient. Although the recruited macrophages persisted, tumor regrowth correlated with a loss of viral activity in the tumor. However, an additional round of MCMV infection further delayed tumor growth, suggesting that tumor growth delay was dependent on active viral infection. Together, our results suggest that MCMV infection delayed the growth of an immunologically cold tumor by recruiting and modulating macrophages in order to promote anti-tumor immune responses.IMPORTANCE Cytomegalovirus (CMV) is an exciting new platform for vaccines and cancer therapy. Although CMV may delay tumor growth in some settings, there is also evidence that CMV may promote cancer development and progression. Thus, defining the impact of CMV on tumors is critical. Using a mouse model of melanoma, we previously found that murine CMV (MCMV) delayed tumor growth and activated tumor-specific immunity although the mechanism was unclear. We now show that MCMV delayed tumor growth through a mechanism that required monocytic phagocytes and a viral chemokine that recruited macrophages to the tumor. Furthermore, MCMV infection altered the functional state of macrophages. Although the effects of MCMV on tumor growth were transient, we found that repeated MCMV injections sustained the antitumor effect, suggesting that active viral infection was needed. Thus, MCMV altered tumor growth by actively recruiting macrophages to the tumor, where they were modulated to promote antitumor immunity.

Keywords: cytomegalovirus; immunomodulation; immunotherapy; macrophages; melanoma.

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Figures

FIG 1
FIG 1
Intratumoral injections of WT MCMV lead to delayed tumor growth. (A) Schematic showing the schedule for intratumoral injections. B16-F0 cells were implanted subcutaneously in the right shaved flank of the animal. Once the tumor reached around 20 mm2, MCMV was injected into the tumor every other day, for a total of three injections corresponding to day 0, day 2, and day 4. (B) C57BL/6J mice were injected i.t. with WT MCMV (n = 12), spread-defective MCMV (n = 8), or PBS (n = 12). Tracings are on a logarithmic scale and show tumor growth in individual animals. Dotted lines indicate the days of i.t. injection. The length and width of the tumor were measured until the tumor reached a size of 100 mm2. The asterisk indicates a tumor that was cleared after the 40-day time point. (C) Kaplan-Meier survival curves were plotted using the day when the tumors reached 100 mm2 as the endpoint. Data were compared with a log rank test. (D) Tumor doubling times for each animal were calculated from day 0 (start of treatment) to sacrifice, excluding mice that cleared their tumor. (E) Representative images show the relative amount of F4/80+ CD11b+ macrophages in each group after treatment. Arrows indicate examples of the cells that stained positive for F4/80 and CD11b in each image. Asterisks in panels C and D denote statistical significance (***, P < 0.001; ****, P < 0.0001). ns, not significant.
FIG 2
FIG 2
Monocytic phagocytes are necessary for tumor growth delay. Mice received control liposomes and WT MCMV (n = 10), control liposomes and PBS (n = 10), clodronate liposomes and WT MCMV (n = 10), or clodronate liposomes and PBS (n = 10). Tracings of tumor growth (A), animal survival (B), and tumor doubling times (C) are shown as described for Fig. 1. (D) Representative images show the relative amount of F4/80+ CD11b+ macrophages in each group after treatment. Arrows indicate examples of the cells that stained positive for F4/80 and CD11b in each image. Asterisks in panels B and C denote statistical significance (**, P < 0.01; ****, P < 0.0001). ns, not significant.
FIG 3
FIG 3
IT-MCMV is ineffective in Rag-knockout mice. Rag KO mice were injected i.t. with spread-defective MCMV (n = 9) or PBS (n = 9) every other day for a total of three injections. Tracings of tumor growth (A), animal survival (B), and tumor doubling times (C) are shown as described for Fig. 1. Asterisks in panels B and C denote statistical significance (*, P < 0.05; **, P < 0.01).
FIG 4
FIG 4
MCK2-mediated recruitment of macrophages is indispensable for tumor growth delay. Tumor-bearing mice were treated i.t. with MCK2mut virus (n = 11), MCK2WT virus (n = 11), or PBS (n = 7). Tracings of tumor growth (A), animal survival (B), and tumor doubling times (C) are shown as described for Fig. 1. (D) Macrophage numbers were measured by immunofluorescent histology for the presence of F4/80+ CD11b+ cells in six representative images of tumors treated with MCK2mut virus (n = 4 tumors), MCK2WT virus (n = 4), and PBS (n = 3). Shown is the density of macrophages per square millimeter of each analyzed image. (E) The frequency of BMDM that were infected by MCK2WT or MCK2mut virus is shown. Four individual wells of macrophages were assessed and the frequency of infected cells was averaged from three images from each well. A two-tailed t test was used to test for differences. Tumor-bearing mice were also treated i.t. with MCK2 KO virus (n = 9), MCK2 WT virus (n = 9), or PBS (n = 6) to confirm initial findings with an independently generated virus. Tracings of tumor growth (F), animal survival (G), and tumor doubling times (H) are shown. Asterisks throughout denote statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). ns, not significant.
FIG 5
FIG 5
MCMV infection repolarizes macrophages to become more M1-like. (A) Polarized and unpolarized BMDMs were infected with MCMV or mock-infected and assessed by qPCR for iNOS (n = 5 independent macrophage cultures), TNF-α (n = 4), IL-1β (n = 3), arginase (Arg; n = 4), and IL-10 (n = 3). Fold change of each transcript relative to untreated M0 macrophages is shown using the calculation 2-(ΔΔCT), and data were compared using a Mann-Whitney test. Asterisks denote statistical significance (**, P < 0.01). ns, not significant. (B) Unsupervised hierarchical clustering of RPPA analyses of infected and uninfected macrophages that were polarized or unpolarized. Shown is a heat map of antibodies that showed significant differences (Benjamini-Hochberg false discovery rate [BHFDR] of <0.05) with or without MCMV infection or between the M1 and M2 polarized states. (C) Monocle pseudotime plot of RPPA data assessing macrophage cell differentiation. Starting from time point 0 (bottom left corner), two distinct lineages appear, separating uninfected M1 and M2 macrophages (M1 to the right and M2 to the top left). In contrast, both MCMV-infected M0 and MCMV-infected M2 macrophages appear on the M1 lineage branch. MCMV-infected M1 macrophages appear at the end of the M1 branch, meaning that there are no differences between the M1 and M1+MCMV samples.
FIG 6
FIG 6
Macrophage-associated transcript and resident macrophage populations are transiently increased following i.t. MCMV injections. (A) Relative expression of the indicated transcripts from whole-tumor homogenate is shown. Tumors treated with WT-MCMV or PBS were harvested on day 5 (WT MCMV and PBS, n = 7), day 8 (WT MCMV, n = 5; PBS, n = 4), and day 11 (WT MCMV, n = 3; PBS, n = 4) after the initial i.t. injection. Data were compared using a Mann-Whitney test. Asterisks denote statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001). ns, not significant. (B) Relative expression of viral transcript is shown as described for panel A. Tumors treated with WT MCMV or PBS were harvested on day 5 (WT MCMV and PBS, n = 4), day 8 (WT MCMV and PBS n = 3), and day 11 (WT-MCMV and PBS, n = 3) after the initial i.t. injection. (C) Concatenated FACS plots show F4/80+ Ly6C+ macrophages on days 5, 8, and 11 after i.t. MCMV or i.t. PBS injection. Plots are concatenated from four samples each and represent populations previously gated on CD45+, NK1.1−, CD3−, CD19−, and CD11b+. (D) Histograms show PD-L1 and MHC-II expression by the indicated macrophage populations from i.t. MCMV-treated tumors shown in panel B, day 8.
FIG 7
FIG 7
Multiple tumor injections recapitulate the original effect of i.t. MCMV therapy and cause a significant increase in survival. Tumor-bearing mice were treated i.t. with three injections of WT MCMV, followed by three injections of PBS (n = 10) or three more injections of WT MCMV (n = 11). The second round of treatment began when tumors surpassed 20 mm2 once again. Tracings of tumor growth (A) and animal survival (B) are shown as described for Fig. 1. Asterisks denote statistical significance (**, P < 0.01). (C) The time to sacrifice for each treatment is shown normalized to the last set of MCMV injections. (D) Using immunofluorescent histology, representative images from the day after the sixth injection show the number of macrophages in the tumor. Arrows indicate examples of the cells that stained positive for F4/80 and CD11b in each image. (E) Macrophage numbers were quantified by counting the number of F4/80+ CD11b+ cells (as described for panel D) in six images from tumors treated with six injections of WT MCMV (n = 3 tumors) or three injections of WT MCMV followed by three injections of PBS (n = 3). Shown is the density of macrophages per square millimeter of each analyzed image. Data were compared using a two-tailed t test, but there was no significant difference.
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