Adt I Androgen Deprivation Therapy

Open access Original research
Targeting a STING agonist to perivascular
J Immunother Cancer: first published as 10.1136/jitc-2024-009368 on 25 July 2024. Downloaded from http://jitc.bmj.com/ on August 8, 2024 by guest. Protected by copyright.
macrophages in prostate tumors delays
resistance to androgen deprivation therapy
Haider Al-janabi,1 Katy Moyes,1 Richard Allen,1 Matthew Fisher,1 Mateus Crespo,2
Bora Gurel,2 Pasquale Rescigno,3 Johann de Bono,2 Harry Nunns,4
Christopher Bailey,5 Anna Junker-Jensen,4 Munitta Muthana,6
Wayne A Phillips ‍ ‍ ,7 Helen B Pearson,8 Mary-Ellen Taplin,9 Janet E Brown,6
Claire E Lewis ‍ ‍ 1
To cite: Al-janabi H, Moyes K, ABSTRACT

Allen R, et al. Targeting a Background Androgen deprivation therapy (ADT) is a
WHAT IS ALREADY KNOWN ON THIS TOPIC
STING agonist to perivascular front-line treatment for prostate cancer. In some men, their ⇒ Androgen deprivation therapy (ADT) is a front-line treat-
macrophages in prostate tumors ment for prostate cancer. However, if not curative, tu-
tumors can become refractory leading to the development
delays resistance to androgen mors often develop resistance and start to regrow and
deprivation therapy. Journal
of castration-resistant prostate cancer (CRPC). This causes
tumors to regrow and metastasize, despite ongoing treatment, metastasize—a condition called castration-resistance
for ImmunoTherapy of Cancer
2024;12:e009368. doi:10.1136/ and impacts negatively on patient survival. ADT is known prostate cancer (CRPC). Prostate cancer is considered
jitc-2024-009368 to stimulate the accumulation of immunosuppressive cells to be an immunologically ‘cold’ tumor type and while
like protumoral tumor-associated macrophages (TAMs), ADT stimulates tumor infiltration by cytotoxic (CD8+) T
► Additional supplemental myeloid-derived suppressor cells and regulatory T cells in cells, they are largely hypofunctional, possibly due to the
material is published online only. prostate tumors, as well as hypofunctional T cells. Protumoral immunosuppressive tumor microenvironment.
To view, please visit the journal TAMs have been shown to accumulate around tumor blood WHAT THIS STUDY ADDS
online (https://doi.org/10.1 136/ vessels during chemotherapy and radiotherapy in other forms
jitc-2024-009368). ⇒ This study is the first to demonstrate that FR-β+ mac-
of cancer, where they drive tumor relapse. Our aim was to
rophages with an immunosuppressive phenotype ac-
see whether such perivascular (PV) TAMs also accumulate
Accepted 10 July 2024 cumulate around blood vessels in mouse and human
in ADT-treated prostate tumors prior to CRPC, and, if so,
prostate tumors during ADT, prior to the onset of CRPC.
whether selectively inducing them to express a potent
Lipid nanoparticles coated with an antibody to FR-β+
immunostimulant, interferon beta (IFNβ), would stimulate
were then used to deliver a STING agonist selectively to
antitumor immunity and delay CRPC.
these perivascular (PV) cells during ADT. This triggered
Methods We used multiplex immunofluorescence to assess
STING signaling and the release of the potent immuno-
the effects of ADT on the distribution and activation status of stimulant, interferon beta, by PV macrophages, which
TAMs, CD8+T cells, CD4+T cells and NK cells in mouse and/ then activated tumor-infiltrating CD8+T cells and de-
or human prostate tumors. We then used antibody-coated, layed the onset of CRPC.
lipid nanoparticles (LNPs) to selectively target a STING agonist,
2′3′-cGAMP (cGAMP), to PV TAMs in mouse prostate tumors HOW THIS STUDY MIGHT AFFECT RESEARCH,
during ADT. PRACTICE OR POLICY
Results TAMs accumulated at high density around blood ⇒ The delivery of an immunostimulant specifically to PV
vessels in response to ADT and expressed markers of a regions of tumors represents a new, more targeted form
protumoral phenotype including folate receptor-beta (FR- of immunotherapy that ensures the activation of T cells
β), MRC1 (CD206), CD169 and VISTA. Additionally, higher as soon as they cross the vasculature into tumors. This
numbers of inactive (PD-1-) CD8+T cells and reduced new approach could be used to extend the treatment
numbers of active (CD69+) NK cells were present in these PV window for ADT in men with prostate cancer. In doing
tumor areas. LNPs coated with an antibody to FR-β selectively so, it would delay/circumvent the need for additional
delivered cGAMP to PV TAMs in ADT-treated tumors, where treatments like radiotherapy and/or or chemotherapy.
© Author(s) (or their they activated STING and upregulated the expression of IFNβ.
employer(s)) 2024. Re-use
This resulted in a marked increase in the density of active
permitted under CC BY-NC. No
CD8+T cells (along with CD4+T cells and NK cells) in PV
INTRODUCTION
commercial re-use. See rights
and permissions. Published by tumor areas, and significantly delayed the onset of CRPC. Prostate cancer is the second most common
BMJ. Antibody depletion of CD8+T cells during LNP administration cancer in men. Androgens are known to
For numbered affiliations see demonstrated the essential role of these cells in delay in CRPC stimulate the progression of this disease via
end of article. induced by LNPs. androgen receptors (ARs) overexpressed on
Conclusion Together, our data indicate that targeting a STING cancer cells. This prompted the development
Correspondence to agonist to PV TAMs could be used to extend the treatment
Dr Claire E Lewis; of drugs that either reduce/ablate androgen
window for ADT in prostate cancer.
Claire.lewis@s heffield.ac.u k synthesis in the testes or reduce AR signaling.
Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 1

Open access
Collectively, these anti-androgens are known as androgen encapsulation in lipid nanoparticles (LNPs), exosomes
deprivation therapy (ADT).1 and bacterial vectors.22
Men with prostate cancer are offered ADT at various In the present study, we show that ADT causes a marked
stages of their disease. Although this initially reduces change in the immune landscape immediately adjacent
tumor burden, some patients develop resistance to ADT, to blood vessels in both mouse and human prostate
a condition called castration resistant prostate cancer tumors just prior to the onset of CRPC. For example, a
(CRPC). Despite the development of various treatment marked increase in the density of protumoral TAMs and
options for CRPC, including androgen/AR signaling naïve (PD-1−) CD8+T cells was seen in these perivascular
inhibitors such as abiraterone and enzalutamide, and (PV) areas, indicating the ability of ADT to promote an
PARP inhibitors, the median survival rate for men with immunosuppressive, PV niche.
metastatic CRPC remains poor at under 3 years.2–4 This We reasoned that the systemic targeting of a STING
highlights the unmet clinical need for novel therapies agonist LNPs to such protumoral, PV TAMs would be
that can prevent/delay the emergence of CRPC. effective due to their close proximity to tumor blood
Mechanistically, the emergence of CRPC has been vessels. So, we synthesized LNPs containing cGAMP and
strongly linked to aberrant AR signaling, defective coated them with an antibody raised against a receptor we
DNA damage/repair pathways, and the intratumoral show is expressed by protumoral PV TAMs in ADT-treated
synthesis of androgens.3 However, AR- independent tumors—folate receptor-beta (FR-β). Following systemic
mechanisms can also facilitate CRPC, including loss of administration of these LNPs to ADT-treated mice bearing
function mutations in the tumor suppressor genes, p53 orthotopic prostate tumors, cGAMP was selectively deliv-
and PTEN, and deregulation of the FGF, TGFβ, RAS/ ered to PV TAMs, triggering STING signaling and their
MAPK, hedgehog and Wnt/β-catenin signaling path- upregulation of IFNβ. This resulted in the activation of
ways. Although therapeutic agents that target one or tumor-infiltrating CD8+T cells (as well as other effectors
more of the above have shown promise in the clinic, like CD4+T cells and NK cells), and delayed the onset of
outcomes remain poor.4 CRPC.
ADT has been shown to increase the frequency of
various immune effector cells in both mouse and human
prostate tumors.5–8 For example, several reports have
shown that tumor infiltration by CD8+ Τ cells increases METHODS
with ADT, although this fails to impact favorably on Orthotopic mouse models of primary prostate cancer
relapse or survival as these cells have reduced cytotoxic Myc-CaP implants
function.5–8 Additionally, ADT has been shown to impair Myc-CaP cells23 stably expressing luciferase were cultured
T cell priming by antigen- presenting cells and T cell in Dulbecco’s Modified Eagle Medium supplemented
expression of cytotoxicity-related genes (granzymes and with 10% fetal bovine serum and 1% penicillin/strepto-
perforins).5 9–11 It is also reported to increase the intra- mycin and were tested regularly for mycoplasma. 50,000
tumoral abundance of various immunosuppressor cell Myc-CaP cells (1:1 PBS:Matrigel) were injected into the
types including tumor-associated macrophages (TAMs), dorsal prostate lobe of male FVB mice (aged 6–8 weeks).
myeloid-derived suppressor cells (MDSCs), and regula- Mice with detectable tumors 7 days later were random-
tory T cells (Tregs).5 12–14 ized into experimental groups and injected subcuta-
Agonists of stimulator of interferon genes (STING)- neously (once on day 7 alone or with a second dose on
signaling pathway in cells are emerging as an exciting form day 14) with either vehicle control (PBS) or degarelix
of immunotherapy for cancer.15 16 These include factors (25 mg/kg) (Ferring Pharmaceuticals, Parsippany, New
that activate STING like cyclic guanosine monophosphate Jersey, USA) (n=5–8/treatment arm). Tumor growth was
(GMP)–AMP synthase (cGAS) or cyclic dinucleotide 2′3′- then monitored every 2–3 days using an IVIS Spectrum
cGAMP (cGAMP).17 Activation of STING in cells causes imaging system (ie, 30 min after injection with 90 mg/kg
them to upregulate type I interferons (IFNs), including d-luciferin).
IFNs αandβ.18 In tumors, the latter have been shown to All Myc-CaP experiments were carried out in compli-
stimulate antitumor immunity via multiple mechanisms ance with UK Home Office Regulations as specified in
including the cross-priming and activation of CD8+T cells the Animals (Scientific Procedures) Act of 1986 and were
by antigen presenting cells, as well as the activation of approved by the Animal Welfare and Ethical Review Body
both CD4+T cells and NK cells.19 20 However, the clinical of the University of Sheffield.
efficacy of STING agonists administered systemically is
compromised by their rapid excretion, low bioavailability, Transgenic Pten-deficient mice
lack of specificity, and adverse, off-target, side effects.21 PBiCre+/−;Ptenfl/fl mice were generated as described previ-
Furthermore, intratumoral administration is limited by ously.24 At 200 days of age, mice were randomly assigned
the accessibility of tumors. To overcome these limita- to sham-castrated or castrated groups. Prostate tumors
tions, a number of delivery systems have been used to were harvested 2 weeks after surgery, snap frozen in liquid
provide protection for STING agonists in the circulation nitrogen, and optimum cutting temperature (OCT)-
and enable them to access tumors. These include their embedded before being cryosectioned.
2 Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

Open access
Transgenic mouse experiments adhered to the guide- prostate or “TURPs”), and five TURPs from patients
lines outlined in the National Health and Medical who received ADT (median treatment time, 7 months).
Research Council Australian Code of Practice for the Three patients in the latter group had draining lymph
Care and Use of Animals for Scientific Purposes and node involvement (ie, early metastasis). Both groups
were approved by the Animal Experimentation Ethics of samples were supplied by The Institute of Cancer
Committee at the Peter MacCallum Cancer Centre. Research, London. These sections were used to assess
T cell subsets relative to CD31+ blood vessels.
Quantitative immunofluorescence staining of mouse prostate
tumors Quantitative immunofluorescence staining of human prostate
Cryosections of mouse prostate tumors were fixed with ice- tumors
cold acetone for 10 min, blocked with 5% goat serum and Antigen retrieval was performed using a pressure cooker
10% mouse FcR blocking solution before being incubated in conjunction with Dako 10X retrieval solution (S1699).
for 40 min with primary antibodies; F4/80 (BIO-RAD, Subsequently, the sections were blocked to reduce non-
1:100), FR-β (BioLegend, 1:100), CD169 (BioLegend, specific binding using 10% goat serum and 1% TBS (at
1:100), VISTA (BioLegend, 1:100), CD206 (MRC1) room temperature for 30 min.).
(BioLegend, 1:100), CD31 (BioLegend, 1:100), CD8 For tumor group 1, sections were incubated in primary
(BioLegend, 1:100), PD-1 (BioLegend, 1:100), phopho- antibodies diluted in 1% bovine serum albumen (BSA)
STING (ThermoFisher 1:100), NK-1.1 (Biolegend 1:100), overnight at 4°C, then washed x3 in TBS, followed by
CD69 (Biolegend 1:100), CD4 (Biolegend 1:100), and secondary antibodies at room temperature for 1 hour
IFNbeta (Invitrogen, 1:100). Primary rabbit antibodies and DAPI for 3 min, before washing and mounting. The
were detected using goat anti-rabbit (IgG) Alexa Fluor primary antibodies used were a mouse monoclonal (IgG3)
555 (Fisher Scientific 1:400). All sections were counter- anti-human CD68 (Dako, Clone PG-M1 1:200), a mouse
stained with 50 ng/mL DAPI solution before washing and monoclonal (IgG1) anti-CD31 (Antibodies.com, Clone
mounting. JC/70A 1:100) and a rabbit polyclonal anti-FR-β (Abcam
A Nikon A1 confocal microscope was used to capture five 1:300). The negative controls for these primary anti-
randomly selected images/tumor (×20 magnification). bodies were species, isotype and concentration-matched
The acquired images were subsequently analyzed using antibodies. The secondary antibodies were a goat anti-
QuPath (V.0.4.3) or ImageJ. For analysis of cell density mouse IgG3- Alexa Fluor 488 (Fisher Scientific 1:200),
relative to blood vessels, cells <15 µm from CD31+blood a goat anti-mouse IgG1-Alexa Fluor 647 (ThermoFisher
vessels were defined as PV. These were counted in each 1:200) and a goat anti-rabbit IgG-Alexa Fluor 555 (Fisher
regions of interest (ROI) and divided by its vessel area Scientific 1:100).
to estimate the PV density/ROI. Cells>15 µm from For tumor group 2, the “MultiOmyx” procedure was
CD31+blood vessels were defined as non-PV, the density used to detect CD8, PD-1 and CD31 as described by us
of which was calculated by dividing cell numbers in these previously25 in untreated and ADT-treated tumors. The
areas of a given ROI, by the total non-PV area of that ROI. antibodies used were mouse anti-human CD8 (Dako),
a rabbit anti-human PD-1 (Abcam), and a mouse anti-
Human primary prostate tumors human CD31/PECAM- 1 (Cell Signaling). All three
Sections were cut from localized, human prostate tumors were conjugated to fluorophores and diluted with 3%
removed from men in the following two groups (supple- (wt/vol) BSA (to working concentrations optimized
mental Table): previously.25 They were applied to sections for 1 hour
1. Matched pretreatment biopsies and post- treatment, at RT, then washed in PBS and high-resolution images
radical prostatectomies (RPs) from 20 patients who collected from 20 ROIs across viable tumor areas using
received neoadjuvant ADT for 6 months at the Dana- a ×20 objective on an INCell analyzer 2200 microscope
Farber Cancer Institute, Boston, USA. Additionally, (GE Healthcare Life Sciences). An AI-based, advanced
pathological staging of prostatectomy samples (ie, af- analytics platform, proprietary to NeoGenomics Labs,
ter ADT) enabled us to divide these into “responder” called “NeoLYTX”, was used to quantify and analyze
or “non- responder” (NR) groups. Responders were subsets of PD-1−CD8+ T cells and PD-1+CD8+ T cells, and
defined as patients with residual prostate cancer of CD31+blood vessels in PV (<15 µm from CD31+blood
≤5 mm, T stage=0–2, after ADT. Whereas NRs had tu- vessels) and non-PV (>15 µm from CD31+blood vessels)
mors that were T stage ≥3 a after ADT (and so were areas of tumors.
starting to regrow and show signs of CRPC). There was
no regional lymph node involvement in either group. Generation of LNPs to selectively target PV TAMs in vivo
These sections were used to assess the coexpression LNPs containing the STING (STimulator of IFN Genes)
of CD68 and FR-β in PV versus non-PV tumor areas agonist, cGAMP, or an inert version of this molecule
in matched tumor samples before and after ADT. An and coated with one of two antibodies, a rat anti-mouse
antibody to CD31 was also used to label blood vessels. FR-β or a rat IgG2a (isotype matched), control antibody
2. Six “untreated” tumors (ie, not given ADT; four pros- (both from BioLegend) were synthesized by LipExoGen
tatectomies and two transurethral resections of the Biotech (as described in online supplemental figure 6A).

Open access
For this, 20 mg/mL D-Lin-MC3-DMA (MC3, MedChe- 2’3-cGAMP and (9) degarelix plus LNPs with no antibody
mExpress) in ethanol, was mixed 1:1 (v/v) with 1,2-dist coating but containing active 2’3-cGAMP.
earoyl-sn-glycero-3-phosphocholine (DSPC, Avanti Polar A further two groups of mice were also included—to
Lipids), cholesterol (Chol, Avanti Polar Lipids), 1,2-dist examine the role of CD8+T cells in mediating the effects of
earoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(p FR-β-antibody coated LNPs containing active cGAMP on
olyethyleneglycol)- 2000] (DSPE-PEG2000, Avanti Polar the onset of CRPC after ADT. For these, mice were admin-
Lipids) and OG488 DHPE (AAT Bioquest) in ethanol. istered i.p. injections of 200 µg of anti-CD8 antibody (or
The molar ratios of the components for the base formula- an isotype control IgG, both supplied by BioXcell). These
tion were as follows: MC3/Chol/DSPC/DSPE-PEG2000/ started 2 days before inoculation with Myc-CaP-LUC cells,
DHPE-OG488 (50/38.5/9.8/1.5/0.2, mol/mol). cGAMP and then proceded every 4 days throughout the course of
(cGAMP, InvivoGen, tlrl-nacga23) or its inactive control tumor growth.
(InvivoGen, tlrl- nagpap) were dissolved in UltraPure Mice were monitored daily for tumor growth using
water (Invitrogen) and diluted in acetate buffer, pH luminometry and well- being (including body weight)
4. LNPs were synthesized using a 3:1 aqueous:organic every 2–3 days. Tumors were snap frozen prior to being
ratio and subsequently washed in UltraPure water using embedded in OCT compound for frozen sectioning.
Amicon centrifugal columns (100 kDa). The encapsu-
lation efficiency of cGAMP (or its inactive control) was Flow cytometry
determined by HPLC. Myc-CaP cells were detached from 6- well plates using
Prior to the functionalization of the LNPs with anti- trypsin/EDTA, washed and resuspended in the following
bodies, 1 mol% of DSPE-PEG2000 was postinserted into primary antibodies: a rat monoclonal (IgG2a) anti-mouse
LNPs. Fc-specific labeling (ie, to ensure antibody attach- FR-β (BioLegend,1:100) or a sheep polyclonal anti-mouse
ment to LNPs in the correct orientation for binding FR-α (R&D Systems, 1:100) for 45–60 min. Cells were
to FR-β on cells) was achieved by performing a click then washed twice and resuspended in FACS buffer. Flow
reaction between the DSPE- PEG2000- DBCO (Broad- cytometry was performed using the BD LSR II flow cytom-
Pharm) and terminal GlcNAz residues on the antibody eter and data processed using FlowJo Software.
carbohydrate domain. To obtain the latter, the SiteClick
Antibody Azido Modification Kit (Invitrogen) was used Statistical analysis
to replace terminal galactose residues on the N-linked All data shown are means±SEMs. Dots on jitter plots repre-
sugars in the Fc region with the azide-containing sugar sent values for individual tumors. All data were analyzed
GalNAz, which is reactive towards DBCO through strain- using GraphPad Prism V.8.02 software. Data analysis was
promoted alkyne- azide cycloaddition. The lipidated conducted blind and statistical analysis performed using
antibodies in DPBS(1X) were postinserted into the the Mann-Whitney U-test with p values of <0.05 consid-
LNPs according to the methods described by Swart et ered to be significant.
al.26 Finally, an additional 2 mol% DSPE-PEG2000 was
postinserted into the LNPs via thin film hydration to
minimize non-specific cellular uptake. The inclusion in RESULTS
LNPs of a phospholipid labeled on the head group with ADT induces PV accumulation of protumoral TAMs in mouse
the bright, green- fluorescent fluorinated fluorescein and human prostate tumors
dye (also called Oregon Green 488), “DHPE-OG488”, The effects of ADT on the distribution and phenoptype
will enable LNP uptake/distribution in tumors to be of TAMs were investigated just prior to CRPC in the
tracked by in vivo. immunocompetent Myc-CaP model. It caused an initial
To test the effects of LNPs in vivo, mice received either reduction in tumor burden within the first 7 days of
a subcutaneous (s.c) injection of PBS or degarelix in PBS treatment but then started to regrow (ie, acquire CRPC)
(“ADT”, Ferring Pharmaceuticals, Parsippany, New Jersey, 7–10 days post-ADT treatment (figure 1A). Sections from
USA); alone 7 days after inoculation (5–8 mice/group), tumors harvested on day 17 from both control and ADT-
then divided into the following groups (with LNPs admin- treated mice were coimmunofluorescently stained for
istered by s.c. injection every 2 days starting 2 days after PBS the macrophage marker F4/80 and the endothelial cell
or ADT): (1) control—that is, PBS, alone (no LNPs); (2) marker CD31. While both PV and non-PV F4/80+TAMs
PBS plus FR-β-antibody coated LNPs containing inactive were increased by ADT relative to the control group
(control) cGAMP (called “LNP(C)” in some figures); (3) (figure 1B,C), a significantly greater increase was seen in
PBS plus FRβ-antibody-coated plus LNPs containing active PV TAMs. Elevated PV F4/80+TAMs were also observed
cGAMP (called “LNP(E)” in some figures); (4) degarelix prior to the acquisition of CRPC in a Pten-deficient trans-
alone (no LNPs); (5) degarelix plus FRβ-antibody coated genic mouse model of prostate cancer (PBiCre+/−;Ptenfl/
fl 23
LNPs containing inactive cGAMP; (6) degarelix plus ) 2 weeks postsurgical castration, compared with sham
FR-β-antibody coated LNPs containing active cGAMP; castration (online supplemental file 4).
(7) degarelix plus control rat IgG- antibody coated To determine if ADT also alters macrophage pheno-
LNPs containing inactive 2’3-cGAMP; (8) degarelix plus type, we interrogated the phenotype of F4/80+TAMs
control rat IgG-antibody coated LNPs containing active in PV and non- PV areas post- ADT using a panel of

Open access
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.

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well-characterized markers of protumoral macrophages, FR-β+CD68+ TAMs to also be higher in NRs than Rs
including folate receptor beta (FR-β), CD169 (SIGLEC1), after ADT (online supplemental file 5). The non-PV
V-domain immunoglobulin suppressor of T cell activation density of these TAMs was also higher in NRs than
(VISTA) and MRC1 (CD206), (figure 1D,E and online Rs before ADT but this was significantly lower than
supplemental file 2).27–30 Strikingly, the density of F4/80+ in PV areas (online supplemental file 5). These data
TAMs coexpressing each of the tumor- promoting cell confirm the abundance of protumoral (FR-β+) TAMs
surface macrophage markers analyzed in PV regions were in PV areas of human prostate tumors after ADT, espe-
significantly upregulated on ADT relative to the control. cially those entering CRPC (ie, NRs).
An increase in TAMs expressing these markers was also
observed in non-PV regions but was significantly lower ADT alters the activation status of various immune effectors
relative to than in PV regions (figure 1D,E and online in PV areas of tumors
supplemental file 2). Collectively, these findings indicate Given that protumoral TAMs were observed to increase
that ADT promotes the accumulation of PV protumor in PV areas on ADT, we reasoned that this might lead
TAMs. to (or coincide with) changes in effectors cells with
Given that both the densities and proportions of PV cytotoxic potential (CD4+and CD8+ T cells, and NK
F4/80+TAMs expressing FR-β, CD169, VISTA or MRC1 cells). To address this, we assessed the density and
were virtually identical (online supplemental file 2), we activation status of these immune effector cells in PV
investigated whether the same PV TAMs coexpressed and non-PV areas of control and ADT-treated Myc-CaP
these markers. Multiplex immunofluorescence staining tumors
for F4/80, FR-β, CD169 and VISTA confirmed that these We show that CD8+T cell density dramatically
markers were expressed by the same TAM subset. This increases significantly in PV areas after ADT (this was
showed that ADT treatment induces a subset of TAMs also observed in non-PV regions although in a signifi-
with a distinct protumoral phenotype in Myc-CaP tumors cantly smaller CD8+T cell population) (figure 2A).
at the onset of CRPC, with significantly higher frequency Interestingly, our analysis of PD-1-expression by CD8+T
in PV areas (online supplemental file 3). cells revealed that the majority (70%) of CD8+T cells
To establish if the observed induction of protumoral accumulating at PV regions on ADT lacked this acti-
PV TAMs was specific to androgen deprivation in the vation marker and so were antigen-naïve (figure 2B).
Myc- CaP model, immunofluorescence staining was While CD8+T cells tended toward a higher density
performed to colocalize F4/80 and MRC1 in primary in PV than non-PV areas of both untreated and ADT-
prostate tumors from PBiCre+;Ptenfl/fl mice, 2 weeks post- treated tumors (figure 2C, right panel), a significant
surgical castration. A similar increase in the PV density of increase in PD-1 negative CD8+T cells was evident in
MRC1+F4/80+TAMs was observed (online supplemental PV (but not non-PV) areas after ADT (figure 2D),
file 4). PV F4/80+TAMs also expressed FR-β following resembling the Myc-CaP model. Together, these data
castration (online supplemental file 4). indicate that ADT causes naive CD8+T cells to accu-
To investigate the clinical relevance of the above mulate in PV areas.
findings, we first examined matching human prostate Analysis of CD4+T cells in Myc-CaP prostate tumors
tumor specimens collected before and after ADT for the showed that CD4+T cells also principally accumulate
human macrophage marker, CD68 and CD31. Immuno- in PV areas of both control and ADT-treated tumors
fluorescence analysis revealed that while the density of (online supplemental figure 5A). However, in both
CD68+TAMs was significantly higher in PV than non-PV PV and non-PV areas, the density and proportion of
areas before and after ADT, their PV density increased CD4+T cells expressing PD- 1 (approximately 50%
further after ADT (figure 1F,G). in all groups) did not change during ADT (online
The density of PV and non-PV FR-β+CD68+TAMs was supplemental figure 5B). So, ADT did not alter the
significantly higher after ADT than before, but this effect distribution or activation status of CD4+T cells in Myc-
of ADT was significantly greater in PV areas (figure 1H&I). CaP tumors.
We then investigated whether CD68+TAM distri- NK cells were identified using the antibody, NK1.1,
bution correlated with tumor responses to ADT by and their activation status investigated using CD69, an
dividing patients who received this treatment for established marker of activation in NK cells (online
6 months into those that showed increased tumor supplemental figure 5C,D). NK cells were more
growth during ADT (ie, were “NRs”) or did not grow frequent in PV than non-PV areas of both control and
(ie, were responders, “Rs”) (online supplemental file ADT- treated Myc- CaP tumors (online supplemental
5). There were no differences in CD68+TAMs between file 5C, right panel) and were almost all CD69 positive
Non-Rs and Rs, before and after ADT. While the FR-β+ (ie, active). A similar trend was observed in non-PV
subset of CD68+TAMs showed a similar PV location areas, however, the density of NK cells was signifi-
to TAMs labeled for CD68 alone (ie, both before and cantly lower in these regions. Of note, differences in
after ADT), the density of PV FR-β+CD68+ TAMs was the PV density of CD69+NK cells in ADT-treated versus
significantly higher for NRs than R’s before ADT. control tumors failed to reach significance (p=0.056)
There was a non- significant (p=0.07) trend for PV (online supplemental figure 5D).

Open access
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 immu-
nofluorescence 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 supple-
mental 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).
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
Figure 2 ADT stimulates the PV accumulation of PD-1- are shown in figure 3B (and selected groups are shown
CD8+T cells in mouse (Myc-CaP) (A, B) and human (C,
separately to facilitate comparisons in figure 3C–F). A
D) prostate tumors. (A, B). (A) Representative fluorescence
images showing the presence of mainly PD-1- CD8+T single dose of ADT was shown to have a similar effect on
cells in PV areas of ADT-treated Myc-CaP (A) and human the growth of Myc-CaP tumors as two ADT doses, indi-
(C) prostate tumors (left panels in both). (A, C) Yellow cating the onset of CRPC within 7 days of ADT in this
arrows=PD-1+CD8+T cells, orange arrows=PD-1-CD8+T model (figures 1A,3C). The various control LNP groups
cells. ADT stimulates the PV accumulation of CD8+T cells showed no effect on tumor growth in the presence or
(A, C, right panels), which are mainly PD-1- (B, D left panels). absence of ADT (online supplemental file 7). In contrast,
The majority of CD8+T cells lack expression of PD-1 across
LNPs coated with FR-β antibody and containing active
tumors, which increases further after ADT (B, D, right panels).
(NPV=non-PV). Data are presented as means±SEMs.
cGAMP significantly delayed the onset of CRPC after
Fluorescence images in A, C are from ADT-treated tumors. ADT (figure 3D). This effect was found to be dependent
*p<0.05, **p<0.01, ***p<0.001. Magnification bars=20 µm. on CD8+T cells as it was abolished by antibody depletion
ADT, androgen deprivation therapy; PV, perivascular. of these effectors in tumors. Alternatively, an isotype-
matched control IgG had no effect on the onset of CRPC
FR-β-targeted LNPs selectively target the STING agonist, after these LNPs (figure 3E).
cGAMP, to PV TAMs in ADT-treated Myc-CaP tumors, and delay Immunofluorescence staining showed that LNPs
CRPC coated with FR-β antibody and containing active cGAMP
To take advantage of these immune-activating functions (“LNPs(E)”) were taken up mainly by PV F4/80+TAMs
of the cGAMP-STING signaling pathway,18 19 we gener- rather than non-PV TAMs or F4/80 cells in PV or non-PV
ated LNPs containing cGAMP and targeted these to PV areas (figure 4A). PV TAMs bearing LNPs were FR-β+
TAMs in ADT-treated tumors by coating them with an (figure 4B–D) and FR-β antibody-coating of LNPs was
antibody to FR-β. The two main aims of this part of the essential for their uptake by PV TAMs. Only<5% of PV
study were to investigate whether FR-β-targeted LNPs F4/80+cells took up LNPs when they had either a control

Open access
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.

Open access
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.

Open access
IgG or no IgG on their surface. This is supported by the PV PD-1+CD4+ T cells was significantly increased during
fact that neither of these two LNP groups (with or without ADT by LNPs(E), but not LNPs(C) (figure 6D,E).
cGAMP) delayed the start of CRPC (figure 3B and online As mentioned previously, the density of NK cells is
supplemental file 7). higher in PV than non-PV areas of both control and ADT-
Immunofluorescence analysis revealed that LNP+- treated Myc-CaP tumors, and they mainly express CD69
FR-β+F4/80+TAMs display p- STING in response to in this location (online supplemental file 6). This was not
LNPs(E) treatment (figure 4E). This was not seen with altered when LNPs(E) or LNPs(C) were administered
either FR-β+ antibody-coated LNPs containing inactive with ADT (figure 7), but the trend toward a lower density
cGAMP (“LNPs(C)”) or LNPs coated with control IgG of activated PV NK cells after ADT alone (online supple-
(data are not shown), indicating that the cGAMP-STING mental file 6, left panel) was reversed by LNPs(E) but not
pathway was successfully activated only by LNPs(E) LNPs(C) (figure 7D).
treatment. The above findings indicate that when LNPs containing
The effect of cGAMP activation of STING on expres- cGAMP are targeted to PV FR-β+ TAMs, it results in their
sion of IFNβ by PV FR-β+ TAMs was then demonstrated. STING activation and increased IFNβ release. While this
The density of LNP+TAMs expressing immunoreactive activated a number of immune effectors in the PV niche,
IFNβ was significantly higher in PV than non-PV areas of the resultant delay in the onset of CRPC after ADT was
LNPs(E)-treated tumors. Indeed, very few IFNβ+LNP+ mediated mainly by CD8+T cells.
TAMswere present in non-PV areas of LNPs(E)-treated Finally, it was important to confirm that LNPs(E) do
tumors (figure 4F). Interestingly, IFNβ was detected not target FR-β+macrophages residing in healthy tissues.
beyond PV TAMs indicating the release of this cytokine We, therefore, examined the effects of LNP exposure on
by PV FR-β+ TAMs and subsequent uptake by neighboring a tissue known to contain FR-β+ macrophages the liver
cells (figure 4G). This accords well with the finding that (online supplemental file 9). FR-β was detected in>80%
many cell types in tumors express receptors for type I of F4/80+Kupffer cells (online supplemental file 9).
IFNs.31 This increase in tumor IFNβ levels after LNPs(E) Despite this, only 20% of these cells took up LNPs or
treatment was not observed with FR-β antibody-coated expressed IFNβ (online supplemental file 9). When the
extent of IFNβ immunoreactivity by all cells was exam-
LNPs bearing the inactive form of cGAMP (“LNPs(C)”)
ined in the liver, only 15%–20% of all nucleated cells
(figure 4H,I), illustrating that LNP(E) treatment is
contained this cytokine (online supplemental file 9).
specific and reliant on cGAMP-STING pathway activity.
This matched the proportion of cells in the liver found to
We then examined the effect of elevated IFNβ (a known
be FR-β+ F4/80+Kupffer cells (online supplemental file
immunostimulant) on the density, distribution and acti-
9) and indicated that little, if any, IFNβ was taken up by
vation status of CD8+T cells, CD4+T cells and NK cells. As
other cell types in the liver.
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
DISCUSSION
of FR-β-antibody coated LNPs with LNPs(E) nor LNPs(C)
ADT is a mainstay treatment for prostate cancer but the
with ADT altered this ADT-induced PV accumulation of development of CRPC limits its long-term efficacy in both
CD8+T cells (figure 5A,B). However, ADT plus LNPs(E) the neoadjuvant and adjuvant settings.1 32 In the current
increased the density and proportion of PV PD-1+CD8+ study, we show that ADT induces the PV accumulation
T cells (ie, reversed the induction by ADT alone of PV of protumoral (FR-β+MRC1+CD169+VISTA+) TAMs in
PD-1-CD8+T cells—see figure 2B). This induction of both human and mouse prostate tumors prior to the
active (PD-1+) CD8+T cells by LNPs(E) occurred only in onset of CRPC. Interestingly, TAMs with a similar pheno-
PV areas of ADT-treated tumors and did not occur with type accumulate in PV areas of mouse tumors during
LNPs(C) (figure 5C). Finally, we immunostained sections chemotherapy and promote tumor relapse.31 33 34 Further
with antibodies against PD-1, LAG3 (a marker of T cell studies are now needed to investigate a possible causal
exhaustion) and CD8 to investigate the functional status link between such PV cells and tumor regrowth in CRPC.
of PV CD8+T cells after LNP treatment. Fully active T cells We also show that ADT causes naïve (PD-1-) CD8+T
were described as CD8+PD-1+LAG3 and exhausted ones cells to gather in PV sites of prostate tumors suggesting
as CD8+PD-1+LAG3+. Figure 5F shows that both PV and their selective recruitment and/or suppression/reten-
non-PV PD-1+CD8+ T cells in the “ADT plus LNPs(E)” tion in these areas. PV TAMs are known to be immuno-
group were predominantly LAG3- (ie, fully active). suppressive27 31 33 so they may inhibit neighboring T cells.
Figure 6A,B shows that CD4+T cells are mainly PV in Indeed, a number of recent studies suggest this may be
Myc-CaP tumors and remain so after ADT. There was a the case. For example, Bao et al35 revealed a close cell-
non- significant trend toward this PV accumulation of to-
cell interaction between a subset of FR-β+MRC1+
CD4+T cells being increased further by the administra- TAMs and inactive (mainly PD-1-) CD8+T cells in human
tion LNPs(E), but not LNPs(C) with ADT. Neither form colorectal carcinomas. Furthermore, depletion of FR-β+
of LNP altered the proportion of CD4+T cells expressing TAMs expressing a transcriptional profile typical of PV
the activation marker, PD-1, in PV areas but the density of TAMs (ie, mrc1, Lyve1, Hmox1,Il10 and stab1) in a mouse

Open access
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.

Open access
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.
model of ovarian cancer increased the frequency and human breast tumors, and suppress T cells and NK cells,
activation of ascitic CD8+T cells.36 possibly via the release of PGE2, ROS and IL-1.40 It is not
VISTA expression by PV TAMs in ADT-treated tumors known whether CD169+PV TAMs do so in ADT-treated
is likely to contribute to the inactive status of PV CD8+T tumors and whether this contributes to the resistance
cells as it is known to bind to receptors/ligands on T cells of primary tumors to ADT. Interestingly, a recent study
and suppress their functions.37 38 This could include their showed that CD169+macrophages promote resistance to
activation by dendritic cells–cells known to be abundant the anti-androgen, enzalutamide (an AR antagonist) in
in PV tumor areas of mouse tumors.39 The coexpression of mouse (prostate) bone metastases.41
CD169 by PV TAMs also suggests an immunosuppressive The increase in PV naïve CD8+T cells seen after ADT
phenotype. A recent study revealed that CD169+TAMs in our studies could also have been due, in part, to
colocalize with T cells (along with regulatory T cells) in thymus enlargement, a known side effect of ADT.42 This

Open access
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.
enhances thymic release of naïve T cells into the circu- LNPs coated with an antibody to FR-β were used to
lation,43 which could lead to more then being recruited deliver the STING agonist, cGAMP, to PV TAMs. In
into tumors. However, thymic release of naïve CD4+T mouse prostate tumors, these LNPs were selectively
cells also occurs during ADT,44 but these cells were not taken up by PV TAMs after ADT, where they released
increased in ADT-treated mouse tumors, suggesting that
cGAMP, activated STING signaling, and released IFNβ.
other mechanisms are involved in the selective increase
The PV niche is an ideal location for this targeted form
in naïve CD8+T cells in PV areas.
The main aim of our study was to see whether “re-ed- of immunotherapy as IFNβ has been shown to stimu-
ucating” PV TAMs to enable them to release a potent late the functions of a number of immune cell types
immunostimulant, IFNβ, reverses the suppressive present in this site in tumors. For example, it stimu-
effects of ADT on tumor-infiltrating T cells. To do this, lates antigen presentation by DCs to naïve T cells,45

Open access
as well as the proliferation and effector functions of Funding This project was funded by a grant from Prostate Cancer-UK to CL and
T cells and NK cells.46 Type I IFNs can also decrease JB (RIA16-ST2-022). HP is supported by a CRUK Career Development award
(#A27894).
the immunosuppressive functions of MDSCs and Tregs
Competing interests No, there are no competing interests.
and promote the expression of antitumor phenotype
of TAMs.47 So, although the antitumor effects of our Patient consent for publication Not applicable.
LNP therapy could be multifaceted in ADT- treated Ethics approval This study involves human participants and the prostate cancer
tumors, our CD8 depletion study showed that these samples provided by the Institute of Cancer Research and Royal Marsden Hospital
in London: patients provided written, informed consent, and the ethics committee
cells play an essential role as mediators of our LNP was the Royal Marsden Hospital Ethics Review Committee (reference no. 04/
approach. IFNβ is known to stimulate the release of Q0801/60). For the second set of prostate cancer samples (provided by the Dana
various potent chemokines for T cells (eg, CXCL9, 10 Farber Cancer Centre, USA), the name of the ethics committee was the Dana-Farber
Cancer Center IRB (reference no. 01-045). Participants gave informed consent to
and 11).48–50 The latter are highly likely to have been
participate in the study before taking part.
involved in the recruitment and activation of CD8+T
Provenance and peer review Not commissioned; externally peer reviewed.
cells seen in tumors after LNP- induction of tumor
IFNβ levels. Data availability statement Data are available on reasonable request. Further
information and requests for data and/or resources/reagents should be directed
To date, clinical trials using such agents (ie, immune to—and will be fulfilled by—the lead contact, CL (claire.lewis@sheffield.ac.uk).
checkpoint inhibitors, vaccines and CAR-T cells) have Supplemental material This content has been supplied by the author(s). It has
shown limited efficacy in prostate cancer.51 Our studies not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been
suggest that such targeted LNPs could be used to enhance peer-reviewed. Any opinions or recommendations discussed are solely those
the efficacy of immunotherapies that require functional of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and
responsibility arising from any reliance placed on the content. Where the content
CD8+T cells in this disease. includes any translated material, BMJ does not warrant the accuracy and reliability
The FR-β-targeted delivery of cGAMP in LNPs to PV of the translations (including but not limited to local regulations, clinical guidelines,
TAMs—and downstream IFNβ—could help circumvent terminology, drug names and drug dosages), and is not responsible for any error
the serious side effects recorded when non-encapsulated and/or omissions arising from translation and adaptation or otherwise.
cGAMP or type I IFNs are injected into the systemic Open access This is an open access article distributed in accordance with the
Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which
circulation. Indeed, no deleterious effects were seen in
permits others to distribute, remix, adapt, build upon this work non-commercially,
the livers of mice injected with cGAMP-containing LNPs and license their derivative works on different terms, provided the original work is
despite the presence of FR-β-expressing Kupffer cells. The properly cited, appropriate credit is given, any changes made indicated, and the use
presence of LNPs and expression of IFNβ were detectable is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
in only a small subset (20%) of these cells with no signs of ORCID iDs
IFNβ uptake by other cell types. This accords well with the Wayne A Phillips http://orcid.org/0000-0002-7961-638X
finding that the expression of receptors for type I IFNs Claire E Lewis http://orcid.org/0000-0003-1938-9530
like IFNβ (IFNARs1 and 2) is negligible in the liver.52
Taken together, our studies show that LNP delivery
of cGAMP to PV TAMs in ADT-treated prostate tumors
increases antitumor immunity and delays CR. If repro- REFERENCES
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Adt I Androgen Deprivation Therapy

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Open access Original research

Targeting a STING agonist to perivascular

To cite: Al-­janabi H, Moyes K, ABSTRACT

Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 1

2 Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 3

4 Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 5

6 Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

could selectively deliver cGAMP to PV TAMs in ADT-­

Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 7

8 Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 9

10 Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 11

12 Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 13

14 Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

Al-­janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 15

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Open access Original research

To cite: Al-janabi H, Moyes K, ABSTRACT

Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 1

2 Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 3

4 Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 5

6 Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

could selectively deliver cGAMP to PV TAMs in ADT-

Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 7

8 Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 9

10 Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 11

12 Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 13

14 Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368

Al-janabi H, et al. J Immunother Cancer 2024;12:e009368. doi:10.1136/jitc-2024-009368 15