ADT induces PV accumulation of protumoral TAMs in mouse and human prostate tumors

The effects of ADT on the distribution and phenoptype of TAMs were investigated just prior to CRPC in the immunocompetent Myc-CaP model. It caused an initial reduction in tumor burden within the first 7 days of treatment but then started to regrow (ie, acquire CRPC) 7–10 days post-ADT treatment (figure 1A). Sections from tumors harvested on day 17 from both control and ADT-treated mice were coimmunofluorescently stained for the macrophage marker F4/80 and the endothelial cell marker CD31. While both PV and non-PV F4/80+TAMs were increased by ADT relative to the control group (figure 1B,C), a significantly greater increase was seen in PV TAMs. Elevated PV F4/80+TAMs were also observed prior to the acquisition of CRPC in a Pten-deficient transgenic mouse model of prostate cancer (PBiCre+/−;Ptenfl/fl)23 2 weeks postsurgical castration, compared with sham castration (online supplemental file 4).

Figure 1

ADT stimulates the PV accumulation of FR-β+ TAMs in mouse (Myc-CaP) and localized, human prostate tumors. (A) Two phases of tumor response to the LHRH antagonist, degarelix, in Myc-CaP tumors: an initial, hormone sensitive (HS) period of tumor growth inhibition followed by the start of castration resistance (CR) when tumors start to regrow. In Myc-CaP tumors (B–E), immunofluorescence staining shows that the density of both PV F4/80+TAMs (B, C)—and the FR-β+ subset of these cells (D, E) increased by the start of CR in ADT-treated tumors. Similar changes occurred in non-PV tumor areas but to a lesser extent than in PV areas. The proportion of F4/80+TAMs expressing FR-β also increased in PV and non-PV areas at this time. Immunofluorescence staining of matched human prostate tumors (F, H) sampled before and after ADT showed that ADT increased the PV density of PV CD68+ tumors (G) and the CD68+TAM subset expressing FR-β (I, left panel). The proportion of CD68+TAMs expressing FR-β rose in PV and non-PV tumor areas after ADT (I, right panel). (NPV=non-PV). Data are presented as means±SEMs. All fluorescence images are of ADT-treated tumors (except the top one in panel B). *p<0.05, **p<0.01, ***p<0.001. Magnification bars=50 µm. ADT, androgen deprivation therapy; LHRH, Luteinising hormone-releasing hormone; PV, perivascular; TAM, tumor-associated macrophage.

To determine if ADT also alters macrophage phenotype, we interrogated the phenotype of F4/80+TAMs in PV and non-PV areas post-ADT using a panel of well-characterized markers of protumoral macrophages, including folate receptor beta (FR-β), CD169 (SIGLEC1), V-domain immunoglobulin suppressor of T cell activation (VISTA) and MRC1 (CD206), (figure 1D,E and online supplemental file 2).27–30 Strikingly, the density of F4/80+ TAMs coexpressing each of the tumor-promoting cell surface macrophage markers analyzed in PV regions were significantly upregulated on ADT relative to the control. An increase in TAMs expressing these markers was also observed in non-PV regions but was significantly lower relative to than in PV regions (figure 1D,E and online supplemental file 2). Collectively, these findings indicate that ADT promotes the accumulation of PV protumor TAMs.

Given that both the densities and proportions of PV F4/80+TAMs expressing FR-β, CD169, VISTA or MRC1 were virtually identical (online supplemental file 2), we investigated whether the same PV TAMs coexpressed these markers. Multiplex immunofluorescence staining for F4/80, FR-β, CD169 and VISTA confirmed that these markers were expressed by the same TAM subset. This showed that ADT treatment induces a subset of TAMs with a distinct protumoral phenotype in Myc-CaP tumors at the onset of CRPC, with significantly higher frequency in PV areas (online supplemental file 3).

To establish if the observed induction of protumoral PV TAMs was specific to androgen deprivation in the Myc-CaP model, immunofluorescence staining was performed to colocalize F4/80 and MRC1 in primary prostate tumors from PBiCre+;Ptenfl/fl mice, 2 weeks postsurgical castration. A similar increase in the PV density of MRC1+F4/80+TAMs was observed (online supplemental file 4). PV F4/80+TAMs also expressed FR-β following castration (online supplemental file 4).

To investigate the clinical relevance of the above findings, we first examined matching human prostate tumor specimens collected before and after ADT for the human macrophage marker, CD68 and CD31. Immunofluorescence analysis revealed that while the density of CD68+TAMs was significantly higher in PV than non-PV areas before and after ADT, their PV density increased further after ADT (figure 1F,G).

The density of PV and non-PV FR-β+CD68+TAMs was significantly higher after ADT than before, but this effect of ADT was significantly greater in PV areas (figure 1H&I).

We then investigated whether CD68+TAM distribution correlated with tumor responses to ADT by dividing patients who received this treatment for 6 months into those that showed increased tumor growth during ADT (ie, were “NRs”) or did not grow (ie, were responders, “Rs”) (online supplemental file 5). There were no differences in CD68+TAMs between Non-Rs and Rs, before and after ADT. While the FR-β+ subset of CD68+TAMs showed a similar PV location to TAMs labeled for CD68 alone (ie, both before and after ADT), the density of PV FR-β+CD68+ TAMs was significantly higher for NRs than R’s before ADT. There was a non-significant (p=0.07) trend for PV FR-β+CD68+ TAMs to also be higher in NRs than Rs after ADT (online supplemental file 5). The non-PV density of these TAMs was also higher in NRs than Rs before ADT but this was significantly lower than in PV areas (online supplemental file 5). These data confirm the abundance of protumoral (FR-β+) TAMs in PV areas of human prostate tumors after ADT, especially those entering CRPC (ie, NRs).

ADT alters the activation status of various immune effectors in PV areas of tumors

Given that protumoral TAMs were observed to increase in PV areas on ADT, we reasoned that this might lead to (or coincide with) changes in effectors cells with cytotoxic potential (CD4+and CD8+ T cells, and NK cells). To address this, we assessed the density and activation status of these immune effector cells in PV and non-PV areas of control and ADT-treated Myc-CaP tumors

We show that CD8+T cell density dramatically increases significantly in PV areas after ADT (this was also observed in non-PV regions although in a significantly smaller CD8+T cell population) (figure 2A). Interestingly, our analysis of PD-1-expression by CD8+T cells revealed that the majority (70%) of CD8+T cells accumulating at PV regions on ADT lacked this activation marker and so were antigen-naïve (figure 2B).

Figure 2

ADT stimulates the PV accumulation of PD-1-CD8+T cells in mouse (Myc-CaP) (A, B) and human (C, D) prostate tumors. (A, B). (A) Representative fluorescence images showing the presence of mainly PD-1- CD8+T cells in PV areas of ADT-treated Myc-CaP (A) and human (C) prostate tumors (left panels in both). (A, C) Yellow arrows=PD-1+CD8+T cells, orange arrows=PD-1-CD8+T cells. ADT stimulates the PV accumulation of CD8+T cells (A, C, right panels), which are mainly PD-1- (B, D left panels). The majority of CD8+T cells lack expression of PD-1 across tumors, which increases further after ADT (B, D, right panels). (NPV=non-PV). Data are presented as means±SEMs. Fluorescence images in A, C are from ADT-treated tumors. *p<0.05, **p<0.01, ***p<0.001. Magnification bars=20 µm. ADT, androgen deprivation therapy; PV, perivascular.

While CD8+T cells tended toward a higher density in PV than non-PV areas of both untreated and ADT-treated tumors (figure 2C, right panel), a significant increase in PD-1 negative CD8+T cells was evident in PV (but not non-PV) areas after ADT (figure 2D), resembling the Myc-CaP model. Together, these data indicate that ADT causes naive CD8+T cells to accumulate in PV areas.

Analysis of CD4+T cells in Myc-CaP prostate tumors showed that CD4+T cells also principally accumulate in PV areas of both control and ADT-treated tumors (online supplemental figure 5A). However, in both PV and non-PV areas, the density and proportion of CD4+T cells expressing PD-1 (approximately 50% in all groups) did not change during ADT (online supplemental figure 5B). So, ADT did not alter the distribution or activation status of CD4+T cells in Myc-CaP tumors.

NK cells were identified using the antibody, NK1.1, and their activation status investigated using CD69, an established marker of activation in NK cells (online supplemental figure 5C,D). NK cells were more frequent in PV than non-PV areas of both control and ADT-treated Myc-CaP tumors (online supplemental file 5C, right panel) and were almost all CD69 positive (ie, active). A similar trend was observed in non-PV areas, however, the density of NK cells was significantly lower in these regions. Of note, differences in the PV density of CD69+NK cells in ADT-treated versus control tumors failed to reach significance (p=0.056) (online supplemental figure 5D).

FR-β-targeted LNPs selectively target the STING agonist, cGAMP, to PV TAMs in ADT-treated Myc-CaP tumors, and delay CRPC

To take advantage of these immune-activating functions of the cGAMP-STING signaling pathway,18 19 we generated LNPs containing cGAMP and targeted these to PV TAMs in ADT-treated tumors by coating them with an antibody to FR-β. The two main aims of this part of the study were to investigate whether FR-β-targeted LNPs could selectively deliver cGAMP to PV TAMs in ADT-treated tumors and stimulate them to express IFNβ, and whether this would stimulate antitumor immunity and delay the onset of CRPC.

As a prelude to the LNP in vivo experiment, we first confirmed that the FR-β antibody used to coat LNPs did not bind to a related molecule, FR-α (known to be expressed by Myc-CaP cells). Flow cytometry and immunofluorescence staining using a specific FR-α antibody alongside our FR-β antibody confirmed that Myc-CaP cells express FR-α but not FR-β, and that the opposite was the case for TAMs in Myc-CaP tumors (online supplemental figure 6B,C).

Multiple, novel formulations of LNPs were synthesized containing either an active or inactive form of cGAMP and coated with either an anti-mouse FR-β antibody or a control IgG (figure 3A; see method for LNP synthesis in online supplemental file 7).

Figure 3

LNPs coated with FR-β-antibody target the STING agonist, cGAMP, to PV TAMs and delay CR in ADT-treated Myc-CaP tumors. (A) Design of LNPs used in vivo. The Fc regions of either a FR-β antibody or a control IgG were attached to LNPs containing either an active cGAMP or an inactive version of this (“cGAMP Ctrl”). (B) Tumor growth in mice administered either PBS or ADT alone, or these followed by administration every 2 days of the various forms of LNP listed. (C and D) Various key groups have been selected from (B) and shown separately (for clarity). (C) Tumor growth curves in response to PBS alone (control) versus a single dose of ADT or (D) PBS alone (control), ADT alone, ADT plus FR-β antibody-coated LNPs containing either cGAMP or cGAMP Ctrl. (E) Effect of in vivo administration of (i) an antibody against CD8 or (ii) an isotype-matched control IgG2b on tumor-infiltrating CD8+T cells after ADT plus FR-β antibody-coated LNPs containing cGAMP (magnification bar=50 µm). (iii) Tumor growth curves showing the effect of depleting CD8+T cells on tumor responses to ADT plus by FR-β antibody-coated LNPs containing cGAMP. Data are presented as means±SEMs. *p<0.001 (comparing tumor sizes at sacrifice). ADT, androgen deprivation therapy; LNP, lipid nanoparticle; TAM, tumor-associated macrophage.

Mice-bearing orthotopic Myc-CaP tumors were then administered either PBS alone (the vehicle for ADT) or a single dose of ADT alone on day seven after implantation (“ADT” group). Two days later, separate groups of control or ADT-treated mice were administered the various forms of LNPs listed in figure 3B (see box). Controls included LNPs coated with either no antibody or a control rat IgG instead of the FR-β antibody, and others containing an inactive form of cGAMP (“cGAMP Ctrl”) rather than active cGAMP. LNP injections were continued every 2 days until day 22, and tumor growth was assessed at regular intervals. None of the LNP groups appeared to have deleterious effects on the mice in terms of their eating/drinking behavior, overall health and body weight (online supplemental file 8).

The effects of these various treatments on tumor growth are shown in figure 3B (and selected groups are shown separately to facilitate comparisons in figure 3C–F). A single dose of ADT was shown to have a similar effect on the growth of Myc-CaP tumors as two ADT doses, indicating the onset of CRPC within 7 days of ADT in this model (figures 1A,3C). The various control LNP groups showed no effect on tumor growth in the presence or absence of ADT (online supplemental file 7). In contrast, LNPs coated with FR-β antibody and containing active cGAMP significantly delayed the onset of CRPC after ADT (figure 3D). This effect was found to be dependent on CD8+T cells as it was abolished by antibody depletion of these effectors in tumors. Alternatively, an isotype-matched control IgG had no effect on the onset of CRPC after these LNPs (figure 3E).

Immunofluorescence staining showed that LNPs coated with FR-β antibody and containing active cGAMP (“LNPs(E)”) were taken up mainly by PV F4/80+TAMs rather than non-PV TAMs or F4/80 cells in PV or non-PV areas (figure 4A). PV TAMs bearing LNPs were FR-β+ (figure 4B–D) and FR-β antibody-coating of LNPs was essential for their uptake by PV TAMs. Only<5% of PV F4/80+cells took up LNPs when they had either a control IgG or no IgG on their surface. This is supported by the fact that neither of these two LNP groups (with or without cGAMP) delayed the start of CRPC (figure 3B and online supplemental file 7).

Figure 4

Selective delivery of cGAMP to PV FR-β+ TAMs in Myc-CaP tumors results in STING activation and upregulation of IFNβ. Following the administration of ADT plus LNPs: (A). The proportion of cells in PV and non-PV areas bearing LNPs that were F4/80− vs F4/80+. (B) Fluorescently labeled LNPs colocalized with PV FR-β+F4/80+TAMs. (C, D) FR-β+F4/80+TAMs bearing LNPs were only present in PV areas. (E) When LNPs bearing active cGAMP (LNPs(E)) were administered, the expression of active phosphorylated STING (P-STING) could be detected in LNP+FR-β+F4/80+TAMs. This was accompanied by a significant increase in IFNβ detection PV LNP+F4/80+TAMs (ie, in the LNPs(E) group) (E). In this group, IFNβ detection was only detectable in F4/80+TAMs in PV not NPV areas, (F) but often extended beyond LNP+cells, indicating its possible release and uptake by other cells in tumors (G). This did not occur when mice were injected with LNPs bearing inactive cGAMP. (NPV=non-PV). Data are presented as means±SEMs. *p<0.05, **p<0.01, ***p<0.001. Magnification bars=50 µm. ADT, androgen deprivation therapy; LNP, lipid nanoparticle.

Immunofluorescence analysis revealed that LNP+FR-β+F4/80+TAMs display p-STING in response to LNPs(E) treatment (figure 4E). This was not seen with either FR-β+ antibody-coated LNPs containing inactive cGAMP (“LNPs(C)”) or LNPs coated with control IgG (data are not shown), indicating that the cGAMP-STING pathway was successfully activated only by LNPs(E) treatment.

The effect of cGAMP activation of STING on expression of IFNβ by PV FR-β+ TAMs was then demonstrated. The density of LNP+TAMs expressing immunoreactive IFNβ was significantly higher in PV than non-PV areas of LNPs(E)-treated tumors. Indeed, very few IFNβ+LNP+ TAMswere present in non-PV areas of LNPs(E)-treated tumors (figure 4F). Interestingly, IFNβ was detected beyond PV TAMs indicating the release of this cytokine by PV FR-β+ TAMs and subsequent uptake by neighboring cells (figure 4G). This accords well with the finding that many cell types in tumors express receptors for type I IFNs.31 This increase in tumor IFNβ levels after LNPs(E) treatment was not observed with FR-β antibody-coated LNPs bearing the inactive form of cGAMP (“LNPs(C)”) (figure 4H,I), illustrating that LNP(E) treatment is specific and reliant on cGAMP-STING pathway activity.

We then examined the effect of elevated IFNβ (a known immunostimulant) on the density, distribution and activation status of CD8+T cells, CD4+T cells and NK cells. As shown in figure 2A (after 2 doses of ADT), a single dose of ADT alone in the LNP experiment resulted in a significant increase in PV CD8+T cells. Neither the coadministration of FR-β-antibody coated LNPs with LNPs(E) nor LNPs(C) with ADT altered this ADT-induced PV accumulation of CD8+T cells (figure 5A,B). However, ADT plus LNPs(E) increased the density and proportion of PV PD-1+CD8+ T cells (ie, reversed the induction by ADT alone of PV PD-1-CD8+T cells—see figure 2B). This induction of active (PD-1+) CD8+T cells by LNPs(E) occurred only in PV areas of ADT-treated tumors and did not occur with LNPs(C) (figure 5C). Finally, we immunostained sections with antibodies against PD-1, LAG3 (a marker of T cell exhaustion) and CD8 to investigate the functional status of PV CD8+T cells after LNP treatment. Fully active T cells were described as CD8+PD-1+LAG3 and exhausted ones as CD8+PD-1+LAG3+. Figure 5F shows that both PV and non-PV PD-1+CD8+ T cells in the “ADT plus LNPs(E)” group were predominantly LAG3- (ie, fully active).

Figure 5

Selective delivery of cGAMP to PV FR-β+ TAMs in Myc-CaP tumors reverses the effect of ADT on the activation status of CD8+T cells in Myc-CaP tumors. (A) Representative fluorescence image showing CD8+T cells in a vascularized area of a tumor treated with ADT plus FR-β antibody-coated LNPs containing active cGAMP (“LNPs(E)”). (B) ADT increased the PV accumulation of CD8+T cells (a change that was unaffected by coadministration with FR-β antibody-coated LNPs containing either inactive cGAMP (“LNPs(C)”) or LNPs(E)). (C) Representative fluorescence image showing colocalization of PD-1 and CD8 in a vascularized area of a tumor treated with ADT plus LNPs(E). (D, E) ADT administered with LNPs(E) reversed the PV accumulation of inactive (PD-1-) CD8+T cells induced by ADT alone, and led to an increase in both the PV density (D) and proportion (E) of CD8+T cells expressing PD-1. (F) The majority of PD-1+CD8+ T cells did not express the exhaustion marker, LAG3 following treatment with ADT plus LNPs(E). (NPV=non-PV). Data are presented as means±SEMs. *p<0.05, **p<0.01, ***p<0.001. Magnification bars=50 µm. ADT, androgen deprivation therapy; LNP, lipid nanoparticle.

Figure 6A,B shows that CD4+T cells are mainly PV in Myc-CaP tumors and remain so after ADT. There was a non-significant trend toward this PV accumulation of CD4+T cells being increased further by the administration LNPs(E), but not LNPs(C) with ADT. Neither form of LNP altered the proportion of CD4+T cells expressing the activation marker, PD-1, in PV areas but the density of PV PD-1+CD4+ T cells was significantly increased during ADT by LNPs(E), but not LNPs(C) (figure 6D,E).

Figure 6

Selective delivery of cGAMP to PV FR-β+ TAMs in Myc-CaP tumors increased the PV accumulation of PD-1+CD4+ T cells in Myc-CaP tumors. (A) Representative fluorescence image showing CD4+T cells in a vascularized area of a tumor treated with ADT plus FR-β antibody-coated LNPs containing active cGAMP (“LNPs(E)”). (B) CD4+T cells were mainly PV in PBS-treated tumors and this was unaffected by ADT alone or coadministration of ADT with FR-β antibody-coated LNPs containing either inactive cGAMP (“LNPs(C)”) or LNPs(E). (C) Representative fluorescence image showing colocalization of PD-1 and CD4 in a vascularized area of a tumor treated with ADT plus FR-β antibody-coated LNPs containing active cGAMP. (D, E) ADT administered with LNPs(E) resulted in the PV accumulation of PD-1+CD4+ T cells. The proportion of CD4+T cells expressing PD-1 remained unaltered by this treatment. (NPV=non-PV). Data are presented as means±SEMs. *p<0.05, ***p<0.001. Magnification bars=50 µm. ADT, androgen deprivation therapy; LNP, androgen deprivation therapy.

As mentioned previously, the density of NK cells is higher in PV than non-PV areas of both control and ADT-treated Myc-CaP tumors, and they mainly express CD69 in this location (online supplemental file 6). This was not altered when LNPs(E) or LNPs(C) were administered with ADT (figure 7), but the trend toward a lower density of activated PV NK cells after ADT alone (online supplemental file 6, left panel) was reversed by LNPs(E) but not LNPs(C) (figure 7D).

Figure 7

Selective delivery of cGAMP to PV FR-β+ TAMs in Myc-CaP tumors increases the PV density of active NK cells in Myc-CaP tumors. (A) Representative fluorescence image showing NK1.1+ NK cells in a vascularized area of a tumor treated with ADT plus FR-β antibody-coated LNPs containing active cGAMP (“LNPs (E)”). (B) NK cells were mainly PV in PBS-treated tumors and this was unaffected by ADT alone. Coadministration of ADT with LNPs (E) increased both the non-PV and PV density of NK cells compared with ADT alone or ADT plus FR-β antibody-coated LNPs containing inactive cGAMP (“LNPs (C)”). (C) Representative fluorescence image showing colocalization of CD69 and NK1.1 in a vascularized area of a tumor treated with ADT plus LNPs (E). (D, E) ADT administered with LNPs(E) increased the PV density of CD69+ (ie, activated) NK cells compared with ADT alone or ADT+LNPs (C). The majority of PV NK cells expressed CD69 in PBS-treated tumors. This did not change after ADT, with or without LNPs. (NPV=non-PV). Data are presented as means±SEMs. *p<0.05, **p<0.01,***p<0.001. Magnification bars=20 µm. ADT, androgen deprivation therapy; LNP, androgen deprivation therapy.

The above findings indicate that when LNPs containing cGAMP are targeted to PV FR-β+ TAMs, it results in their STING activation and increased IFNβ release. While this activated a number of immune effectors in the PV niche, the resultant delay in the onset of CRPC after ADT was mediated mainly by CD8+T cells.

Finally, it was important to confirm that LNPs(E) do not target FR-β+macrophages residing in healthy tissues. We, therefore, examined the effects of LNP exposure on a tissue known to contain FR-β+ macrophages the liver (online supplemental file 9). FR-β was detected in>80% of F4/80+Kupffer cells (online supplemental file 9). Despite this, only 20% of these cells took up LNPs or expressed IFNβ (online supplemental file 9). When the extent of IFNβ immunoreactivity by all cells was examined in the liver, only 15%–20% of all nucleated cells contained this cytokine (online supplemental file 9). This matched the proportion of cells in the liver found to be FR-β+ F4/80+Kupffer cells (online supplemental file 9) and indicated that little, if any, IFNβ was taken up by other cell types in the liver.