Tumoral immune suppression by macrophages expressing fibroblast activation protein-α and heme oxygenase-1

Abstract

The depletion of tumor stromal cells that are marked by their expression of the membrane protein fibroblast activation protein-α (FAP) overcomes immune suppression and allows an anticancer cell immune response to control tumor growth. In subcutaneous tumors established with immunogenic Lewis lung carcinoma cells expressing ovalbumin (LL2/OVA), the FAP(+) population is comprised of CD45(+) and CD45(-) cells. In the present study, we further characterize the tumoral FAP(+)/CD45(+) population as a minor subpopulation of F4/80(hi)/CCR2(+)/CD206(+) M2 macrophages. Using bone marrow chimeric mice in which the primate diphtheria toxin receptor is restricted either to the FAP(+)/CD45(+) or to the FAP(+)/CD45(-) subset, we demonstrate by conditionally depleting each subset that both independently contribute to the immune-suppressive tumor microenvironment. A basis for the function of the FAP(+)/CD45(+) subset is shown to be the immune inhibitory enzyme, heme oxygenase-1 (HO-1). The FAP(+)/CD45(+) cells are the major tumoral source of HO-1, and an inhibitor of HO-1, Sn mesoporphyrin, causes the same extent of immune-dependent arrest of LL2/OVA tumor growth as does the depletion of these cells. Because this observation of immune suppression by HO-1 expressed by the FAP(+)/CD45(+) stromal cell is replicated in a transplanted model of pancreatic ductal adenocarcinoma, we conclude that pharmacologically targeting this enzyme may improve cancer immunotherapy.

Figure 1

FAP expression by tumoral CD45⁺ cells marks a subpopulation of F4/80^hi macrophages. A, A representative FACS dot plot of gated CD45⁺ cells from a single cell suspension of an LL2/OVA tumor stained with the additional antibodies shown. B, The proportion of cells in five LL2/OVA tumors that were F4/80^hi and either FAP⁺ or FAP⁻. C, FACS histograms of gated tumoral FAP⁺ and FAP⁻ F4/80^hi cells stained with the specific antibodies shown (open) and isotype controls (Shaded).

Figure 2

The selective depletion of DTR-expressing FAP⁺/CD45⁺ and FAP⁺/CD45⁻ tumoral cells from bone marrow chimeric mice by the administration of DTX. A, Sketch depicting the donor-recipient combinations for generating the chimeric mice. B, The proportions of each of the two subsets of FAP⁺ cells in LL2/OVA tumors in individual bone marrow chimeric mice that had received DTX for three consecutive days. C, The tumor volumes (left) and the calculated percent change in growth (right) of LL2/OVA tumors in the groups of bone marrow chimeric mice that were given DTX for three consecutive days. ns- not significant, * p<0.05, ** p<0.01, *** p<0.001.

Figure 3

Expression of HO-1 by the FAP⁺/F4/80⁺ tumoral macrophages. A, Frozen sections of LL2 tumors from FAP/EGFP BAC Tg mice in which FAP⁺ cells are identified by native EGFP fluorescence (green) were also stained with DAPI (blue) and antibodies specific for F4/80 (white) and HO-1 (red). B, Randomly selected HO-1⁺ cells across multiple sections and fields were evaluated in three separate LL2 tumors for co-expression of FAP/EGFP and F4/80 (>300 total HO-1⁺ events were characterized). C, Relative HO-1 mRNA expression in sub-populations of FACS-sorted tumoral populations from an LL2/OVA tumor.

Figure 4

Effect of inhibiting HO-1 on adaptive immune- and cytokine-dependent regulation of tumor growth. A, C57BL/6 mice bearing established, non-immunogenic LL2 or immunogenic LL2/OVA tumors, and Rag2^−/− mice with LL2/OVA tumors were treated with daily administration of SnMP (25μMol/Kg) or vehicle control, with the arrow denoting the initiation of treatment. B and C, Mice bearing established LL2/OVA tumors were treated with SnMP (25μMol/Kg) or vehicle for 24 hr after which the change in tumor volumes (B) and the expression of TF by CD31⁺ endothelial cells were measured (C). D, Mice bearing LL2/OVA tumors that had been pre-treated with neutralizing anti-TNF-α and anti-IFN-γ antibodies or with isotype control antibody were given SnMP (25μMol/Kg) or vehicle, and tumor volumes were measured 24 hr later. The curves describing tumor growth in (A) were compared for differences using the “CompareGrowthCurves” permutation test. ns- not significant, * p<0.05, ** p<0.01, *** p<0.001.