Integrin CD11b regulates macrophage polarization

We previously reported that the VCAM receptor integrin α4β1 promotes myeloid cell recruitment from the bone marrow to the tumor microenvironment, thereby stimulating immune suppression, angiogenesis and tumor progression2,13,14,15. In contrast to its role in regulating recruitment of myeloid cells to tissues during acute inflammation16,17,18, we found that CD11b (αMβ2), a myeloid cell integrin receptor for ICAM-1 and fibrinogen, does not affect myeloid cell recruitment to tumors, as global deletion of CD11b in Itgam−/− mice has no effect on the number of myeloid cells in circulation or in the number of cell recruited to tumors (Supplementary Figure 1; Supplementary Figure 2a-d). Surprisingly, however, we found that integrin CD11b plays an essential role in regulating macrophage polarization. Itgam−/− macrophages exhibited enhanced immune suppressive gene and protein expression and strongly reduced pro-inflammatory gene and protein expression compared with WT macrophages, whether stimulated under basal, IL-4 or IFNγ/LPS stimulation conditions (Fig. 1a, Supplementary Figure 2e-f). To determine whether CD11b also regulates macrophage polarization in vivo, we isolated and characterized F4/80 + TAMs from LLC tumors grown in Itgam−/− and WT mice. We found that Itgam−/− TAMs also expressed significantly higher levels of mRNAs associated with immune suppression and angiogenesis, such as Arg1, Tgfb, Il10, Il6, and Pdgfb, and significantly lower expression of genes associated with immune stimulation, such as Ifng, Nos2, and Tnfa than did WT TAMs (Fig. 1b, Supplementary Figure 2g).

Fig. 1 CD11b ligation promotes pro-inflammatory macrophage signaling. a–f Relative mRNA expression of pro- and anti-inflammatory cytokines in a bone marrow derived macrophages (BMDM) from WT (white bars) or Itgam−/− (cyan bars) mice (n = 2–8); b tumor associated macrophages (TAM) from WT (white bars) and Itgam−/− (cyan bars) mice bearing LLC lung carcinoma tumors (n = 2–4); c Itgam−/− and Itgam or non-silencing siRNA transfected macrophages (n = 2–4); inset: cell surface expression levels of CD11b in transfected macrophages; d WT macrophages in the presence of non-specific (IgG) or anti-CD11b antibodies (n = 3), e murine macrophages adherent to ICAM-1, VCAM-1 or BSA coated plates (Susp) (n = 3) and f human macrophages adherent to ICAM-1 or BSA coated plates (Susp) (n = 3). g Immunoblotting of phosphoSer536 and total p65 NFκB RelA in WT and Itgam−/− macrophages stimulated with IFNγ + LPS; graph depicts quantification of relative pSer536 expression in WT (white bars) and Itgam−/− (blue bars) BMDM. h, i LLC tumor growth in WT and Itgam-/- mice adoptively transferred with h bone marrow derived and i tumor derived WT or Itgam−/− macrophages. j Relative mRNA expression of cytokines in whole LLC tumors from WT (white bars) and Itgam−/− (cyan bars) mice (n = 3). k LLC lung (n = 17), B16 melanoma (n = 9) and autochthonous PyMT mammary (n = 10–14) tumor weight in WT (black dots) and Itgam−/− (cyan dots) mice. l Tumor weight and volume of LLC tumors grown in WT (black dots) versus Itgam I332G knockin mice (cyan dots) (n = 6–7). Error bars indicate sem. “n” indicates biological replicates. p < 0.05 indicates statistical significance determined by Student’s t-test for a–g and j. and by Anova with Tukey post-hoc testing for h, i, k, l. Source data are provided in Source Data file Full size image

Importantly, transient siRNA-mediated knockdown of CD11b in in vitro cultured macrophages elevated immune suppressive gene expression and decreased immune stimulatory gene expression, effects that are comparable to CD11b deletion (Fig. 1c), indicating that even transient loss of CD11b controls macrophage immune suppressive gene expression. To address whether CD11b expression or function controls macrophage gene expression, we examined the effect of inhibitory CD11b antibodies on macrophage mRNA expression. Blockade of murine macrophage CD11b mediated attachment to ICAM-1-coated substrates by anti-CD11b neutralizing antibodies also induced immune suppressive mRNA expression in macrophages (Fig. 1d). Similarly, adhesion of macrophages to the integrin α4β1 substrate VCAM-1 or loss of attachment by suspension culture promoted murine and human immune suppressive transcription, while attachment to ICAM-1 coated surfaces promoted immune stimulatory transcription (Fig. 1e, f, Supplementary Figure 1h), indicating that ligation of CD11b controls immune stimulatory macrophage transcription.

The loss of pro-inflammatory cytokine expression in Itgam−/− macrophages suggested that CD11b may regulate activation of pro-inflammatory transcription factors, such as NFκB. We found that Itgam−/− macrophages exhibited reduced NFκB serine 536 phosphorylation (an indication of reduced activation19) in response to LPS stimulation compared to WT macrophages, suggesting CD11b plays a role in NFκB activation (Fig. 1g). As other studies have implicated CD11b in the promotion of pro-inflammatory responses of monocytes and dendritic cells through direct interactions of LPS with integrin beta2 extracellular domains20,21, our results indicate that CD11b activation and signaling play key roles in the regulation of macrophage polarization in vitro and in vivo.

Macrophage CD11b regulates tumor growth

Our data indicate that Itgam−/− bone marrow derived and tumor associated macrophages exhibit more immune suppressive transcriptional profiles than WT macrophages. To determine if this difference affects tumor growth, we adoptively transferred WT or Itgam−/− bone marrow derived or tumor associated macrophages with tumor cells into recipient WT or Itgam−/− mice. Previously, we demonstrated that adoptively transferred, immune suppressive BMDM or TAMs can stimulate tumor growth9,10. Remarkably, bone marrow-derived Itgam−/− macrophages (Fig. 1h) as well as tumor-derived Itgam−/− macrophages (Fig. 1i) potently stimulated tumor growth compared with WT macrophages in both WT and Itgam−/− mice. As Itgam−/− macrophages exhibit an immune suppressive transcriptional profile (Fig. 1b), and tumors derived from Itgam−/− mice exhibit an overall immune suppressive transcriptional profile (Fig. 1j), these data suggested that CD11b expression or activation might impact overall tumor growth. Indeed, we found that subcutaneous (LLC), orthotopic (melanoma) and autochthonous (PyMT) tumors grew more aggressively in Itgam−/− than in WT mice (Fig. 1k). As Itgam−/− mice exhibited substantially more CD4 + Foxp3 + T regs and fewer CD8+ T cells in tumors than WT mice (Supplementary Figure 3a-b), our studies support the conclusion that CD11b plays a key role in regulating the overall immune response in tumors.

Prior studies have shown that Isoleucine 332 in the CD11b molecule serves as an allosteric switch controlling the adhesion receptor’s activation and shape22. To determine whether CD11b activation controls tumor development, we generated a constitutively activated CD11b knockin mouse strain (C57BL/6 ITGAMI332G by introducing an I332G point mutation in the murine Itgam gene. I332G knockin mice express normal levels of cell surface CD11b on both monocytes and granulocytes and exhibit normal levels of all blood cell level (Supplementary Figure 3c-d). In vitro adhesion assays with bone marrow derived macrophages from these mice showed that I332G cells express constitutively active CD11b (Supplementary Figure 3e). Importantly, I332G Itgam knockin mice exhibited significantly reduced LLC tumor growth (Fig. 1k). Thus, while CD11b deletion stimulates anti-inflammatory macrophage polarization, inhibits CD8+ T cell recruitment and promotes tumor growth, CD11b activation potently inhibits tumor growth. These studies indicate that macrophage CD11b plays a critical functional role in controlling tumor growth.

Immune suppressive signals inhibit CD11b expression

To determine whether signals associated with the tumor microenvironment can alter CD11b expression and subsequently affect myeloid cell polarization, we evaluated the effect of macrophage media (mCSF-, IL-4- and IFNγ/LPS) on cell surface CD11b expression in bone marrow derived macrophages. While the immune suppressive cytokine IL-4 reduced CD11b expression, the pro-inflammatory stimuli IFNγ/LPS enhanced CD11b surface expression compared to levels expressed on mCSF-stimulated macrophages (Supplementary Figure 4a-b). Additionally, the immune suppressive factor TGFβ, but not IL-10, inhibited CD11b surface expression (Supplementary Figure 4c); TGFβ, IL-4 and tumor cell conditioned medium (TCM) each also suppressed Cd11b mRNA expression (Supplementary Figure 4d). Importantly, TGFβ and TCM reduced CD11b cell surface expression and stimulated immune suppressive transcription while inhibiting immune stimulatory transcription in a manner that was reversed by the TGFβR1 inhibitor SB525334 (Supplementary Figure 4e-g). These data indicate that cytokines such as TGFβ in the tumor microenvironment suppress CD11b expression or activation, thereby promoting immune suppressive macrophage polarization.

Macrophage CD11b regulates blood vessel stability

Macrophages not only control immune responses but also angiogenesis and desmoplasia, by expressing cytokines such as VEGF-A and PDGF-BB, growth factors that regulate endothelial cell and vascular smooth muscles/pericytes during angiogenesis, respectively2. Tumor blood vessels often consist of a single endothelial layer that lacks supporting pericytes or smooth muscle cells; these blood vessels are more numerous in tumors than in normal tissues but are aberrantly formed and poorly perfuse. In contrast, in tumors with high PDGF to VEGF ratios, blood vessels are lined by pericytes, mesenchymal cells that stabilize vessels and promote better tumor perfusion23,24,25,26,27. These tumors grow more rapidly than tumors with lower PDGF to VEGF ratios but also respond better to chemotherapy and immune therapy due to better tumor perfusion24,25,26,27,28,29,30,31,32,33,34,35,36. As Itgam−/− macrophages exhibited high PDGF and low VEGF gene expression (Fig. 1a, b), we examined the patterns of blood vessel development in Itgam−/− and WT tumors. An assessment of vascular patterning in LLC and PyMT tumors from WT and Itgam−/− mice showed that Itgam−/− tumors exhibited fewer, longer blood vessels with wider lumens and fewer branch points/field than WT tumors (Fig. 2a, b; Supplementary Figure 5a). Itgam−/− tumors had more blood vessels that were lined with Desmin+, NG2+, or SMA+ pericytes/smooth muscle cells than did WT tumors (Fig. 2a–c; Supplementary Figure 5b). Accordingly, these vessels were less permeable in Itgam-/- mice than in WT mice, as less intravascular FITC-dextran leaked into the tumor parenchyma (Fig. 2a–d). These data indicate that the integrin CD11b plays a role in controlling blood vessel maturation. We found that gene expression of PDGF-BB but not VEGF-A was strongly enhanced in tumors (Fig. 2e; Supplementary Figure 5c). Importantly, PDGF-BB protein expression was also elevated in Itgam−/− tumors and tumor-derived macrophages compared to WT tumors (Fig. 2f). Together, these data suggest that macrophage CD11b controls tumor vascularization through the constitutive expression of elevated levels of PDGF.

Fig. 2 CD11b suppresses PDGF-BB-dependent neovascularization and tumor growth. a CD31, Desmin, NG2, Smooth muscle actin (SMA) and Dextran immunostaining of blood vessels in LLC tumors from WT (white bars) and Itgam−/− (blue bars) mice (n = 10). b Blood vessel density (vessels/field) and number of vessel branch points/field in LLC tumors from WT (white bars) and Itgam−/− (blue bars) animals (n = 10). c Percent CD31+/SMA+, CD31+/Desmin+, CD31+/NG2+ vessels in tumors from WT (white bars) and Itgam−/− (blue bars) (n = 5). d Ratio of extravascular to intravascular FITC-dextran in LLC tumors from WT (white bars) and Itgam−/− (blue bars) mice (n = 5). e, f Pdgfb and Vegfa e mRNA (n = 3) and f PDGF-BB protein expression (n = 12) in LLC tumors from WT (white bars) and Itgam−/− (blue bars) mice (n = 3). g Left, FITC-Isolectin staining of whole mount retinas from newborn WT (white bars) and Itgam−/− (blue bars) mice. Right, Percent neovascularization at P1, P4, and P9 retinas in WT (white bars) and Itgam-/- (blue bars) mice (n = 5). h Desmin (red) and CD31 (green) immunostaining of LLC tumors from Imatinib and vehicle-treated WT (white bars) and Itgam-/- (blue bars) mice. i Tumor weight, number of blood vessels/field and percent Desmin/CD31 vessels per field from h (n = 10). Bar on micrographs indicates 50 µm. Error bars indicate sem. “n” indicates biological replicates. p < 0.05 indicates statistical significance, as determined by Student’s t-test for b–g and by Anova with Tukey’s post-hoc testing for i. Source data are provided in Source Data file Full size image

In support of these observations on CD11b roles in neovascularization, we found that Itgam−/− mice exhibited a well-developed retinal vascular plexus (Fig. 2g, Isolectin B+, green) at birth (P1) compared with WT mice, which exhibit undeveloped retinal vasculature that expands progressively from postnatal day 1 (P1) to P9. The superficial vascular plexus was more developed in Itgam−/− mice from postnatal day P1 through postnatal day P9 than in WT neonates (Fig. 2g). These results indicate that macrophages and CD11b play key roles in the control normal vascular patterning.

To investigate whether elevated PDGF-BB is responsible for the enhanced vascular maturation and tumor growth in Itgam−/− mice, we treated WT and Itgam−/− mice bearing LLC tumors with imatinib, an inhibitor of the PDGF-BB receptor PDGFR1. Imatinib treatment suppressed the enhanced tumor growth observed in Itgam−/− mice (Fig. 2h, i). It also increased vascular density and suppressed vascular normalization in Itgam−/− mice (Fig. 2h, i). Together these results support the concept that integrin CD11b modulates vascular development through control of PDGF-BB expression.

CD11b regulates Let7a and c-Myc expression

CD11b may control anti-inflammatory macrophage polarization through the activation of transcription factors such as Stat3, which can promote expression of immune suppressive and pro-angiogenic factors such as Arginase 1, Myc, and VEGF37,38,39. Itgam−/− macrophages exhibit constitutively phosphorylated Stat3 (Fig. 3a inset); the high levels of immune suppressive factor expression in Itgam−/− macrophages were reduced to WT macrophage levels by treatment with the Stat3 inhibitor 5,15-DPP (Fig. 3a). Surprisingly, however, Stat3 inhibition did not affect the high levels of Il6 expression observed in Itgam−/− macrophages (Fig. 3a). Importantly, IL-6 can directly activate Stat338. We found that IL-6 promoted the same pattern of immune suppressive polarization in murine and human myeloid cells and macrophages we observed in Itgam-/- macrophages (Fig. 3b). Together, these results suggested that autocrine IL6 may drives the constitutively immune suppressive polarization observed in Itgam−/− macrophages. In support of this concept, Il6 knockdown decreased expression of constitutive Pdgfb expression in Itgam−/− macrophages (Fig. 3c). As TAMs are a major source of Il6 expression in tumors15, these results suggest that CD11b serves as a natural brake on immune suppression in part through control of myeloid cell transcription of Il6.

Fig. 3 CD11b promotes miR-Let7a mediated immune stimulation. a Relative mRNA expression of pro-inflammatory and anti-inflammatory factors in WT (white bars) and Itgam−/− (blue bars) macrophages incubated with and without the Stat3 inhibitor 5,15 DPP; inset, Stat3 phosphorylation in WT and Itgam−/− macrophages (n = 2–3). b Relative mRNA expression of pro-inflammatory and anti-inflammatory factors in IL6-stimulated human (white bars) and murine (cyan bars) bone marrow-derived macrophages and murine total bone marrow derived myeloid cells (blue bars) (n = 3); p < 0.05 with these exceptions: mBMM (Ifng, Il12b); mCD11b+ (Arg1, Pdgfb, Il12b and Il1b); hBMM (Arg1, Ifng, Il1b). c Relative Il6 and Pdgfb mRNA expression in WT and Itgam−/− cells transduced with non-silencing (white bars or Il6 (blue bars) siRNA (n = 3). d Relative Let7a expression in murine macrophages transduced with non-silencing (white bars) or Itgam (blue bars) siRNAs, macrophages incubated with control IgG (white bars) or neutralizing anti-CD11b (blue bars) antibodies, and WT (white bars) or Itgam−/− (blue bars) macrophages (n = 3). e Time course of Let7a (left) and Il6 (right) expression in WT murine CD11b+ cells seeded on ICAM-1 (blue solid line) or maintained in suspension (black dotted line) (n = 3). f Relative expression of miRNA Let7a and Il6 in human macrophages adherent to ICAM-1 (blue) or maintained in suspension (white) (n = 3). g Relative mRNA expression of inflammatory factors in WT and Itgam-/- BMM transduced with control (white bars), pre-miRNA Let7a (cyan bars) or anti-miRNA Let7a (blue bars) (n = 3). h Relative Pdgfb and Vegfa expression in WT BMM transduced with control (white bars) or anti-miRNA Let7a (blue bars) (n = 3). i Time course of c-Myc expression in IL-4 or IFNγ + LPS stimulated WT (black lines) or Itgam−/− (blue lines) macrophages (n = 3). j Time course of c-Myc expression and pSer62myc phosphorylation in WT and Itgam−/− macrophages. k Relative mRNA expression of miRNAs Let7a (white bars), Let7d (blue bars) and Let7f (cyan bars) in basal, IL-4, or IL-4+ c-myc inhibitor treated WT and Itgam−/− macrophages (n = 3). l Relative mRNA expression of Il6, Arg1, and Pdgfb in IL-4 stimulated WT (white bars) and Itgam−/− (blue bars) macrophages treated with (blue bars) or without (white bars) c-Myc inhibitor 10058-F4. Error bars indicate sem. “n” indicates biological replicates. *p < 0.05 indicates statistical significance determined by Student’s t-test for a, d–f, and Anova with Tukey’s post-hoc testing for b, g, k. Source data are provided as a Source Data file Full size image

The Let7 family of microRNAs can controls Il6 expression in tumor and inflammatory cells40,41. microRNAs are non-coding RNAs that modulate gene expression at the post-transcriptional level by interfering with RNA translation or stability and can dramatically impact tumor immune suppression and angiogenesis42,43. We found that miRNA Let7a expression inversely correlated with Il6 expression in murine and human macrophages (Supplementary Figure 6a-b). Therefore, we asked whether loss of CD11b expression in macrophages affects Let7a expression. Let7a expression was ablated in Itgam−/− and Itgam siRNA transduced macrophages and in in the presence of neutralizing CD11b antibodies (Fig. 3d; Supplementary Figure 6c) in a manner that was independent of Lin28, an RNA binding protein that cleaves and inactivates Let7, as Lin28 levels were not affected by CD11b expression or activation (Supplementary Figure 6b, d-e). CD11b ligation by ICAM-1 promoted time-dependent Let7a expression, while inhibiting Il6 expression; conversely, suppression of adhesion inhibited Let7a expression and promoted Il6 expression in both murine and human macrophages (Fig. 3e, f). Importantly, ectopic expression of Let7a miRNA (pre-miRNA) inhibited immune suppressive gene expression and stimulated pro-inflammatory gene expression in Itgam−/− macrophages, while anti-miRNA Let7a stimulated immune suppressive gene expression and inhibited immune stimulatory gene expression in WT macrophages (Fig. 3g). Similar to CD11b ablation (Fig. 1a), anti-miRNA Let7a stimulated Pdgfb expression but had no effect on Vegfa expression (Fig. 3h). Together, these results indicate that CD11b activation promotes miRNA Let7a expression, which in turn inhibits IL6-mediated immune suppressive macrophage gene expression.

c-Myc, a transcription factor that regulates immune suppressive macrophage polarization, binds to the Let7 promoter and suppresses its transcription; interestingly Let7 can also suppress c-Myc expression44,45. We found that c-Myc gene was upregulated in Itgam−/− macrophages compared with WT macrophages (Fig. 3i). c-Myc protein expression and serine 62 phosphorylation, which stabilizes the transcription factor46, were also upregulated in Itgam−/− macrophages compared with WT macrophages (Fig. 3j). We then asked whether inhibition of cMyc function could promote Let7 expression and thereby alter macrophage polarization. Importantly, Let7a, Let7d, and Let7f expression was reduced in Itgam−/− macrophages; however, pharmacological inhibition of c-Myc restored Let7 expression in Itgam−/− macrophages and reversed the increased immune suppressive gene expression exhibited by Itgam−/− macrophages (Fig. 3k, l). Together, these data indicate that integrin CD11b functions to suppress Myc expression and immune suppressive macrophage polarization in a Let7 dependent manner.

Because Let7a inhibits macrophage-mediated Pdgfb expression, we investigated the effect of Let7a expression on neovascularization in vitro and in vivo. Endothelial cells and vascular smooth muscle cells attached to microcarrier beads were cultured in fibrin gels that contained either WT or Itgam−/− macrophages that were transduced with control miRNA, pre-miRNA Let7a, anti-miRNA Let7a or Pdgfb-bb siRNA. Itgam−/− macrophages stimulated sprout elongation that was inhibited by transduction of macrophages with Let7a miRNA or Pdgfb siRNA (Fig. 4a, b; Supplementary Figure 6f). In contrast, expression of anti-miRNA Let7a in WT but not Itgam-/- macrophages stimulated sprout elongation (Fig. 4a, b; Supplementary Figure 6f). Additionally, macrophages transduced with anti-miR Let7a stimulated the formation of mature, pericyte-coated blood vessels in bFGF-saturated Matrigel in vivo (Fig. 4c). Together, these studies show that CD11b controls neovascularization through the regulation of Let7a and subsequent PDGF-BB expression.

Fig. 4 Macrophage microRNA let-7a is required for tumor growth suppression. a, b Endothelial cells and vascular smooth muscle cells attached to microcarrier beads were cultured in fibrin gels containing WT or Itgam−/− BMMs transduced with control miRNA, pre-miRNA Let7a, anti-miRNA Let7a or Pdgf-bb siRNA. a Images b histograms of CD31+ positive vessel length (mm) (n = 10). c CD31 (green) and SMA (red) immunostaining of sections from in vivo cultured bFGF-saturated Matrigel plugs containing BMM transduced with control miR (black bars) or anti-miR Let7a (blue bars); quantification of the percentage of SMA+ vessels per matrigel plug (n = 25). d Schematic and graph of targeted delivery of anti-miR Let7a in animals with LLC tumors; tumor volumes from control anti-miRNA (black line) or anti-miRNA Let-7a (cyan line) treated animals (n = 10). e Let7a expression in cell populations sorted from peripheral blood cell and tumors from control (black bars) and anti-miRNA Let7-treated (cyan bars) animals from d (n = 3). f Relative mRNA expression of inflammatory factors in sorted macrophages from d (n = 3). g Representative images of CD31/Desmin co-localization and FITC-Dextran localization in treated tumors from d. h Quantification of the percentage of CD31/Desmin co-localization (n = 30) and of FITC-dextran leakage into tissues (n = 25). i CD4+ and CD8+ cells/field in tumors from control anti-miR (black bars) or anti-miR let-7a (blue bars) transduced animals (n = 25) scale bars, 40 µm. j Schematic representation of chemotherapeutic treatment in combination with targeted delivery of anti-miR-let7a. k Volumes and endpoint weights of LLC tumors in animals transduced with control anti-miR (black), anti-miR let-7a (blue), control anti-miR /Gemcitabine (green), and anti-miR let7a/Gemcitabine (red) (n = 10). Bar on micrographs indicates 50 µm. Error bars indicate sem. “n” indicates biological replicates. *p < 0.05 indicates statistical significance by Student’s t-test for c–i and by Anova with Tukey’s post-hoc testing for j Source data are provided as a Source Data file Full size image

Myeloid cell Let7a regulates tumor progression

To test the role of Let7a in the regulation of tumor immune suppression and neovascularization, we delivered anti-miR Let7a to tumors in myeloid cell targeted nanoparticles in vivo (Fig. 4d). We found that integrin αvβ3-targeted nanoparticles were specifically taken up by circulating myeloid cells in normal and tumor bearing animals (Supplementary Figure 7a-h). Delivery of anti-miRNA Let7a stimulated LLC tumor growth, comparable to that observed in Itgam−/− mice (Fig. 4d). Although Let7a is expressed in immune and non-immune cells in tumors, we found that delivery of anti-miR Let7a only inhibited Let7a expression in circulating monocytes and in tumor associated macrophages but not in other tumor associated cells (Fig. 4e). Importantly, anti-miRNA Let7a stimulated immune suppressive and pro-angiogenic gene expression and inhibited pro-inflammatory gene expression in tumors compared with controls (Fig. 4f, Supplementary Figure 8a). Anti-miRNA Let7a also stimulated blood vessel normalization in transfected tumors, as vessels were longer, less branched, heavily coated with pericytes and less leaky than vessel from control transfected tumors (Fig. 4g, h). Importantly, anti-Let7a also suppressed CD8+ T cell recruitment to tumors and enhanced CD4+ T cell recruitment to tumors (Fig. 4i). Together, these results indicate that CD11b restrains immune suppression and vascular maturation through its regulation of miRNA Let7a. Prior studies have shown that increased vascular normalization in tumors can improve tumor perfusion and promote responsiveness to therapy23,24,25,26,27,28,29,30,31,32,33,34,35,36. To determine whether the vascular normalization induced by anti-miRNA Let7a might enhance the efficacy of chemotherapy by increasing tumor perfusion, we treated mice bearing LLC tumors with targeted delivery of anti-miRNA Let7a or control miRNA in combination with chemotherapy (gemcitabine) (Fig. 4j). Whereas anti-miRNA Let7a promoted LLC tumor growth, anti-miRNA Let7a combined with gemcitabine substantially reduced tumor growth, consistent with the notion that Let7a inhibition increases accessibility of the tumor to chemotherapy (Fig. 4k). In accordance with these results, we found that gemcitabine treatment of Itgam−/− mice suppressed tumor growth more profoundly than gemcitabine treatment of WT mice (Supplementary Figure 8b). As Itgam−/− exhibited greater perfusion (less vascular leak) than WT mice (Supplementary Figure 8c), these studies indicate that CD11b, through its effects on miRNA Let7a, plays a critical role in regulating tumor immune and vascular responses.

The CD11b agonist LA1 inhibits tumor growth

Our results suggested that targeted pharmacologic activation of CD11b in vivo might repolarize tumor associated macrophages, with subsequent inhibition of tumor immune suppression and tumor growth. We thus investigated the effects of a small molecule agonist of CD11b, leukadherin 1 (LA1)47,48 (Fig. 5a) on macrophage polarization and tumor growth. LA1 stimulated myeloid cell adhesion to ICAM-1 coated substrates in a manner that was inhibited by anti-CD11b-neutralizing antibodies (Fig. 5b). LA1 stimulated macrophage immune response gene expression, illustrated by increases in expression of Il1b, Tnfa, Il12, Nos2, and Ifng mRNAs (Supplementary Figure 9a). As LA1 stimulated Let7a expression and inhibited Pdgfb and Il6 expression (Fig. 5c), these results suggested that LA1 might stimulate pro-inflammatory immune responses that could inhibit tumor growth in vivo. To assess the effects of LA1 on tumor associated macrophages in vivo, tumor associated macrophages were isolated10, treated with LA1 prior and co-implanted with LLC tumor cells. LA1-treated macrophages completely inhibited tumor growth (Fig. 5d, e) even though LA1 had no direct effect on LLC or macrophage viability (Fig. 5e, f). Although LA1 had no effect on CL66-Luc breast tumor cell growth in vitro (Supplementary Figure 9b), LA1 potently reduced tumor growth in syngeneic, orthotopically implanted CL66-Luc breast tumors more effectively than taxol (Fig. 5g). LA1 also synergized with irradiation to suppress CL66-Luc breast tumor growth (Fig. 5h) and suppressed the growth of orthotopic, human MDA-MB-231 mammary xenograft tumors (Fig. 5i). Importantly, LA1 inhibited murine LLC lung tumor growth in WT but not in Itgam−/− mice, indicating that LA1 acts through integrin CD11b to suppress the growth of tumors (Fig. 5j, k).

Fig. 5 Integrin CD11b agonism suppresses tumor growth and promotes survival in mouse models of cancer. a Structure of LA1. b Adhesion of macrophages in the absence or presence of Ca2+Mg2+ (white bars) Mn2+ (black bars), LA1 (cyan bars) or LA1+ neutralizing anti-CD11b (gray bars) (n = 3). c Relative mRNA expression of Let7a, Il16, or Pdgfb in control (black bars) and LA1 (cyan bars)-treated macrophages (n = 3). d Tumor weights 16 days after implantation of LLC cells mixed 1:1 with control-treated (dots), DMSO-treated (triangles) or LA1-treated (diamonds) tumor-derived macrophages (n = 8). e Tumor growth curves as represented by volumes from d: control (black line), vehicle (gray line) and LA1 (red line) (n = 8). f Effect of LA1 on in vitro proliferation of LLC cells and macrophages (n = 4). g Tumor volumes of orthotopic CL66 breast tumors treated with vehicle (black line), Taxol (gray line), LA1 (dark blue line) or LA1 + Taxol (cyan line) (n = 10–15). h Tumor volumes of orthotopic CL66 tumors treated with vehicle (black line), irradiation (gray line) (IR, 20 Gy), LA1 (dark blue line) (2 mg/kg), or LA1 + IR (cyan line) (n = 9). i Tumor volumes of orthotopic human MDA-MB-231 mammary xenografts treated with vehicle (control, black line), Taxol (gray line), or LA1 (blue line) (n = 7). j, k Mean LLC subcutaneous tumor volumes of j WT (black line) and k Itgam-/- (cyan line) mice treated with and without LA1 (n = 6). l Images and quantification of SMA/CD31 expression in blood vessels of control (black bars) and LA1 treated (cyan bars) animals from g, i, and j. Bar on micrographs indicates 50 µm. m Schematic depicting role of CD11b activation in the control of immune stimulation. Error bars indicate sem. “n” indicates biological replicates. *p < 0.5 indicates statistical significance by Student’s t-test c, f; Anova with Tukey post-hoc testing for d–e; unpaired t-test g, i; Mann–Whitney t-test b, j–l; Wilcox test 5 h. Source data are provided as a Source Data file Full size image

As LA1 treatment increased the presence of MHC-II + macrophages, typically considered immune competent, and decreased the presence of CD206+ macrophages, typically considered immune suppressive, in LLC and CL66-Luc tumors (Supplementary Figure 9c-d), our studies suggest that LA1 repolarizes tumor associated macrophages. Accordingly, we found that LA1 inhibited expression of S100A8 and MMP9 in CD11b+ cells in LLC tumors and also inhibited expression of Arginase1, S100A8 and MMP9 in CL66-Luc tumors (Supplementary Figure 9e-f). As these proteins are markers of pro-tumoral macrophages, together these studies indicate that LA1 likely inhibits tumor growth by repolarizing tumor associated macrophages. Indeed, LA1 treatment increased the presence of CD8 + T cells in both LLC and CL66-Luc tumors (Supplementary Figure 10a-c). We also observed that LA1 treatment altered neovascularization in tumors by decreasing the numbers of SMA + blood vessels (Fig. 5l). By enhanced the pro-inflammatory immune profile of tumors and inhibiting vascular normalization in tumors, the small molecule CD11b agonist LA1 significantly altered macrophage polarization, increased CD8+ T cell recruitment to tumors and inhibited tumor progression in mouse models of murine and human cancer.