Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Mar 1.
Published in final edited form as: Radiother Oncol. 2024 Jan 13;192:110091. doi: 10.1016/j.radonc.2024.110091

The radiopharmaceutical radium-223 has immunomodulatory effects in patients and facilitates anti-programmed death receptor-1 therapy in murine models of bone metastatic prostate cancer

Philip J Saylor 1,*, Sergey V Kozin 2,*, Aya Matsui 2, Saveli I Goldberg 2, Shuichi Aoki 2, Kohei Shigeta 2, Emilie Mamessier 2, Matthew R Smith 1, M Dror Michaelson 1, Richard J Lee 1, Dan G Duda 2
PMCID: PMC10905770  NIHMSID: NIHMS1961949  PMID: 38224917

Abstract

Background & purpose:

Radium-223 (Ra223) improves survival in metastatic prostate cancer (mPC), but its impact on systemic immunity is unclear, and biomarkers of response are lacking. We examined markers of immunomodulatory activity during standard clinical Ra223 and studied the impact of Ra223 on response to immune checkpoint inhibition (ICI) in preclinical models.

Materials & methods:

We conducted a single-arm biomarker study of Ra223 in 22 bone mPC patients. We measured circulating immune cell subsets and a panel of cytokines before and during Ra223 therapy and correlated them with overall survival (OS). Using two murine mPC models—orthotopic PtenSmad4-null and TRAMP-C1 grafts in syngeneic immunocompetent mice—we tested the efficacy of combining Ra223 with ICI.

Results:

Above-median level of IL-6 at baseline was associated with a median OS of 358 versus 947 days for below levels; p=0.044, from the log-rank test. Baseline PlGF and PSA inversely correlated with OS (p=0.018 and p=0.037, respectively, from the Cox model). Ra223 treatment was associated with a mild decrease in some peripheral immune cell populations and a shift in the proportion of MDSCs from granulocytic to myeloid. In mice, Ra223 increased the proliferation of CD8+ and CD4+ helper T cells without leading to CD8+ T cell exhaustion in the mPC lesions. In one of the models, combining Ra223 and anti-PD1 antibody significantly prolonged survival, which correlated with increased CD8+ T cell infiltration in tumor tissue.

Conclusion:

The inflammatory cytokine IL-6 and the angiogenic biomarker PlGF at baseline were promising outcome biomarkers after standard Ra223 treatment. In mouse models, Ra223 increased intratumoral CD8+ T cell infiltration and proliferation and could improve OS when combined with anti-PD1 ICI.

Keywords: prostate cancer, immunomodulation, immunotherapy, translational research, immune checkpoint blockade

INTRODUCTION

Ra223 is a radiopharmaceutical approved for the treatment of castration-resistant prostate cancer (CRPC) metastatic to bone and without visceral or bulky lymph node metastases. It produces an OS benefit and a delay in time to the first symptomatic skeletal event (1,2). Despite these benefits, Ra223 does not typically produce PSA responses, and its mechanism of efficacy is not fully understood. Prognostic biomarkers are limited (3), predictive biomarkers are lacking, and the lack of mechanistic understanding limits its rational use in combination regimens.

Systemically delivered Ra223 accumulates in the highly vascularized areas of increased bone turnover (4,5). Ra223 emits alpha particles, distinguishing it from conventional low-linear energy transfer (LET) photon and proton radiotherapies. Such alpha-particle radiation features high LET, causing more double-strand DNA damage and allowing it to potentially overcome hypoxia-mediated radioresistance. This heavy ionization is localized, with the alpha-particle traveling less than 100μm (6). Ra223 is thought to directly irradiate a fraction of cancer cells near its bony deposition and potentially suppress tumor-induced pathologic bone turnover (7).

A key feature of the metastatic prostate cancer (mPC) microenvironment is profound immunosuppression mediated by infiltrating immune cells, such as myeloid-derived suppressor cells (MDSCs) and T-regulatory cells (Tregs), and by cytokines and chemokines, such as IL-6 or SDF1α. These factors counteract effector T-cell activation and expansion (8,9). Our prior studies in patients with recurrent PC showed that elevated levels of plasma IL-6, IL-1β, IL-8, TNFα, and SDF1α correlated with more aggressive disease. Initiation of androgen deprivation therapy (ADT) was associated with lower plasma IL-6, whereas multiple inflammatory markers such as IL-1β, IL-8, and SDF1α remained higher than in the controls (10). These inflammatory factors may reflect resistance to standard treatments such as radiopharmaceuticals and immunotherapies. Little is known about their dynamics during Ra223 or their association with outcome.

Immunosuppression in the tumor microenvironment is thought to account for resistance to immunotherapy. Anti-programmed death receptor (PD)-1 therapy has had limited activity in unselected mPC (11–15). Whether combining Ra223 and ICI can enhance adaptive immune responses is unclear. The specific immunomodulatory effects of systemic Ra223 remain largely unknown, as they may differ from local external beam radiation therapy (EBRT). Irradiation with Ra223 is different from EBRT in radiobiological and spatiotemporal properties. EBRT can have immune-stimulating effects but can also lead to the depletion of circulating lymphocytes and increase immunosuppressive factors such as Tregs and MDSCs (16,17). One study of Ra223 in mPC patients found a decreased mean frequency of PD-1+CD8+ memory T cells in blood but no effects on other immune checkpoint molecules or cytokines (18).

We aimed to address these knowledge gaps through both clinical and preclinical studies. To better understand the systemic effects of Ra223 therapy, we examined immune cell subsets and markers of inflammation and angiogenesis in peripheral blood before and during treatment. We further interrogated the efficacy and biological impact of Ra223 alone and in combination with ICI in preclinical models of bone mPC. The central question for these model systems was whether Ra223 combined with ICI could increase intratumoral T-cell infiltration and improve survival. We sought within these models to compare systemic to local immune-related changes with Ra223, with or without ICI.

MATERIALS AND METHODS

CLINICAL

Patients:

Participants were aged ≥18 years with histologically or cytologically confirmed prostate adenocarcinoma with bone-predominant metastases and castration resistance. The trial was approved by the Dana Farber/Harvard Cancer Center (DF/HCC) institutional review board and registered at Clinicaltrials.gov (NCT02346526).

Treatment:

This was a single-institution, single-arm study. All participants received standard-of-care Ra223 infusions every 4 weeks for up to 6 doses.

Study endpoints:

This prospective exploratory biomarker study was designed to assess inflammatory and angiogenic biomarkers during standard-of-care treatment with Ra223 for bone metastatic CRPC. Blood and imaging data were collected throughout study participation at specified timepoints. OS was the primary clinical endpoint to be correlated with these exploratory biomarkers.

Cellular and molecular blood biomarkers:

Cellular and molecular biomarkers were examined in serial blood samples collected pre-treatment (day 0) and immediately prior to all subsequent Ra223 infusions as previously described (19).

PRECLINICAL

Orthotopic bone mPC models in mice:

We injected PtenSmad4-null PC cells in F1 mixed background (FVB x C57Bl/6) mice (matching the genetic background of the Pten/Smad4-null mice) or in FVB mice; TRAMP-C1 PC cells were injected in C57Bl/6 mice (matching the genetic background of TRAMP mice). When the thickness of the hindlimbs with the tumor reached 6.5 mm (day 0; the normal thickness of hindlimbs is approximately 4 mm), the mice were randomly assigned to treatment.

Treatments:

Ra223 dichloride was provided by Bayer (Cardinal Health, Denver, CO) in 2–3ml aliquots of an overall activity of about 50μCi. Mice received Ra223 diluted in sterile saline before treatment for IV injections into the tail vein or retro-orbitally in a volume of about 150μl at a dose of 55 kBq/kg. Antibodies against PD-1 (aPD1) (BioXCell, Lebanon, NH) were administered i.p. at 10 mg/kg diluted in 100μl sterile PBS. Ra223 was administered once (at day 0), and the antibodies were dosed every 3 days up to a total of 3–5 injections, starting on day 0.

Endpoints and analysis of tumors, blood, and spleen:

Therapeutic efficacy was evaluated by tumor growth inhibition and modulations of mouse survival and secondary lung metastasis. Immune cells were analyzed in the blood, spleen, and tumor tissue 6–10 days after treatment initiation. Changes induced in the mPC microenvironment (vessel structure, hypoxia) were evaluated by immunohistochemistry.

The work was done under protocol #2016N000081 titled, “Radium-223 dichloride with anti-PD1/CTLA-4 immunotherapy in bone metastasis model of prostate carcinoma in mice”, approved by the MGH IACUC.

For additional Methods, see supplement.

RESULTS

CLINICAL

Clinical outcomes and baseline blood parameters within the prospective clinical cohort of Ra223-treated mPC patients

A total of 22 participants were enrolled from 9/2015 to 7/2019. Baseline clinical characteristics are in Table 1. Eighteen patients received at least 3 planned cycles of Ra223 during study participation and, of those, 14 received all 6 planned cycles. Off-study criteria leading to fewer than 6 cycles included clinical cancer progression, clinician discretion, and patient decision; in the present cohort, this was typically due to clinical progression concurrent with progressive disease on imaging. None of the on-study doses were delayed. Dosing weight range was 64.4–110.7kg.

Table 1.

Baseline characteristics for the clinical cohort.

Clinical cohort %
Age median (range) 71 (49–88) -
Total alkaline phosphatase median (range) 117.5 (45–460) -
PSA median (range) 34.6 (1.2–167.7) -
ECOG performance status 0 10 45
1 12 55
Number of bone metastases <6 8 36
6 – 20 7 32
>20 7 32
Current bisphosphonate or denosumab yes 4 18
no 18 82
Prior chemotherapy yes 5 23
no 17 77
Prior abiraterone yes 17 77
no 5 23
Prior enzalutamide yes 5 23
no 17 77
Prior sipuleucel T yes 1 5
no 21 95
Prior lines of overall survival prolonging therapy* 0 1 5
1 17 77
2 4 18
Open in a new tab
*

Note: Androgen receptor signaling inhibitors (e.g., abiraterone or enzalutamide) are together considered a single line of OS prolonging therapy given limited efficacy of subsequent ARSI after one line of such therapy.

Median OS for the cohort was 21.3 months (95% CI: 11.3–36.7), which appeared favorable compared to the 14.0 months observed in the phase III ALSYMPCA study (1). The OS of 8 patients who stopped Ra223 before completing 6 planned cycles was shorter than that of the 14 who received the full course of therapy (median OS 9.4 [95% CI: 2.1–29.2] and 27.3 [95% CI: 13.9–49.7] months, respectively; log-rank p=0.017).

Of the potential circulating biomarkers measured pretreatment, several were correlated with OS. Among plasma molecules examined using a median baseline concentration cut-off, a significant correlation with OS was observed for IL-6 and non-significant trends for PlGF and PSA (Fig. 1A, C, E). Using Cox regression model analysis, baseline circulating PlGF, and PSA showed a negative correlation with OS (Fig. 1D, F).

Figure 1. Baseline biomarkers and overall survival.

Figure 1.

Figure 1.

Figure 1.

Open in a new tab

(A) The regression curve shows a trend toward a negative correlation between baseline IL-6 and survival. (B) Patients with above-median baseline IL-6 had significantly shorter OS. Regression curves with significant negative correlations between PlGF and OS (C) and PSA and OS (E). Kaplan-Meier analyses for those two biomarkers with patients dichotomized by baseline concentrations relative to median demonstrate trends toward longer OS in those with below-median levels for PlGF (D) and PSA (F).

Table S1 summarizes the results of our analyses for the 8 most notable of the 20 circulating molecular and cellular biomarkers. There was no trend for correlation between baseline plasma SDF1α or TGF-β and OS. Moreover, we found no statistically significant correlations with OS for WBCs or circulating immune cell subsets at baseline. There were non-significant trends for a longer OS with lower baseline plasma counts of MDSC subsets and Tregs.

Patients who terminated Ra223 early for study-specific reasons (largely due to clinical progression) had shorter OS than those who received all six treatments. Interestingly, there was no association between early Ra223 termination and higher baseline levels of the studied plasma molecules or cells. We next performed exploratory analyses that excluded the 8 patients who discontinued Ra223 early. For the cohort of 14 patients who completed Ra223 treatment, we found significant correlations between the baseline circulating biomarkers (plasma IL-6 and PlGF, serum PSA, and circulating Tregs and M-MDSCs) and OS (Table S1).

Changes in circulating biomarkers during Ra223 and their correlation with OS

We also performed exploratory analyses of the dynamics of circulating biomarker candidates during Ra223 treatment and their association with the outcome. We measured molecular and cellular biomarkers during treatment and correlated their changes with OS for patients who completed all 6 infusions of Ra223. Fig. 2A-C depicts the changes in circulating IL-6, PlGF, and PSA in these patients dichotomized by median OS. For plasma IL-6 and PlGF and serum PSA, we found increases in the patients with shorter survival. Of note, only plasma PlGF was significantly different at pretreatment in these two sub-groups. In contrast, in mPC patients with OS longer than the median the mean levels of these molecules remained relatively stable.

Figure 2. Dynamics of biomarkers and cell populations during Ra223.

Figure 2.

Figure 2.

Open in a new tab

We measured molecular and cellular biomarkers over the course of Ra223 and correlated their changes with OS for patients who completed all 6 infusions. Longitudinal alterations of IL-6 (A), PlGF (B), and PSA (C) in the blood are shown with patients dichotomized by OS times below and above the median. There was modest on-treatment leukopenia (D) due primarily to decreased concentrations of neutrophils particularly one month after the first Ra223 infusion. Several detailed WBC characteristics determined by flow analysis – Treg (E), ratio CD8/Treg (F), Gr-MDSCs (G) and M-MDSCs (H) are also shown.

For cellular biomarkers, we observed modest on-treatment leukopenia due primarily to decreased neutrophil counts, most pronounced one month after the first Ra223 infusion (Fig. 2D). Lymphocyte counts decreased more gradually while monocyte counts remained unchanged. We observed no trend after dichotomization relative to the median OS for the whole cohort of patients who completed Ra223 treatment. However, we found higher initial levels of Tregs and both MDSC subsets, and further increasing of M-MDSCs in mPC patients with shorter OS. There was also a tendency for a higher CD8/Treg ratio before and during the whole Ra223 treatment in these patients (Fig. 2E-H).

We then tested how biomarker changes correlated with OS for this cohort. Changes in plasma PlGF both early (8 weeks) and late (20 weeks) from baseline correlated with OS, similar to baseline PlGF (Table S2). In contrast, only a late increase of plasma IL-6 and only an early increase of M-MDSCs correlated with shorter OS after Ra223 treatment. The results for these biomarkers are consistent with the associations seen after dichotomization by OS (Fig. 2).

PRECLINICAL

Immunomodulatory effects Ra223 in preclinical models of bone mPC

To explore the potential immune effects of Ra223 both systemically and in tumor tissues, we treated PtenSmad4-null mPC-bearing FVB immunocompetent mice with a single dose (55kBq/kg) of Ra223, sacrificed them after 10 days, and analyzed lymphocyte populations in mPC and spleen tissues by flow cytometry. In tumor tissues, we found a higher frequency of CD8+ T-cell infiltration compared to CD4+ T-cells, including helper T-cells and Tregs, and Ra223 treatment increased the proliferation of both CD8+ and CD4+ helper T-cells (Fig. S1A, B). The majority of CD8+ cells were PD1+ (over 80%), and Ra223 treatment resulted in a trend to a decreased ratio of activated (PD1+Tim3–) versus exhausted (PD1+Tim3+) CD8+ cells (Fig. S1C, D). Flow cytometric analysis of splenocytes revealed no changes in systemic levels of CD4 or CD8 T-cell subsets (Fig. S1E, F).

We then evaluated the effects of Ra223 and aPD1, alone or combined in two aggressively growing bone mPC models. In the PtenSmad4-null mPC model in F1 mice, single-dose Ra223 treatment was ineffective and aPD1 alone demonstrated a trend to increased OS; however, only their combination achieved a statistically significant increase of OS (Fig. 3A). Lung metastases were detectable in approximately 20% of mice at the terminal endpoint in all treatment groups. The tumor growth rate in the TRAMP-C1 mPC model was even faster, and the spontaneous secondary lung metastatic burden was higher than in the PtenSmad4-null model. Neither Ra223 nor aPD1 alone showed significant activity in the TRAMP-C1 mPC model; however, their combination significantly increased median OS (Fig. 3B). This was due to both an additive effect of the agents in delaying tumor growth (Fig. S2A) and a decrease of secondary lung metastatic burden (Fig. 3C & Fig. S2B); thus, aPD1 was a stronger player in this pair.

Figure 3. Anti-tumor efficacy of Ra223 and anti-PD1 antibody (anti-PD1) in bony mPC models.

Figure 3.

Open in a new tab

(A) Kaplan-Meier distributions for time to endpoint after 55 kBq/kg Ra223, 10mg/kg x 3 anti-PD1 or their combination in PtenSmad4-null mPC in F1 (FVBxC57Bl/6) mice (n=19–20 per group, performed in duplicate); *p<0.05 for the effect of combined treatment vs. control. (B) Kaplan-Meier distributions for time to endpoint after 55 kBq/kg Ra223, 10mg/kg x3 anti-PD1 or their combination in TRAMPC1 in C57Bl/6 mice (n=12 per group, performed in duplicate); **p<0.01 for the difference between combined group and control. (C) In these TRAMPC1-bearing mice, Ra223 and anti-PD1 treatment significantly reduced lung metastasis burden measured at the experimental endpoint, *p<0.05; two-way ANOVA shows that aPD1 over significantly affects the result, p=0.024.

Next, we examined CD8+ T-cell infiltration and vascular changes in the microenvironment of TRAMP-C1 bone mPC by IF using tissues collected on day 6 of treatment. We found increased intratumoral infiltration by CD8+ CTLs after Ra223 alone and a further increase after combination therapy (Fig. 4A). Transcriptomic analysis using RNAseq data via GSEA found significant enrichment in pathways related to immune response, including IFN signaling in tumors from Ra223-treated groups (Figs. S3-S5 and Table S1). Neither treatment significantly changed tumor vessel density, but the mPCs from mice treated with aPD1 overall showed an increased faction of mature vessels covered by α-SMA-positive perivascular cells (Fig. S6A-D). This potential “normalizing” effect was associated with a trend for a decrease in tissue hypoxia in the combination group (Fig. S6E). Notably, flow cytometry analysis of the spleen and blood cells did not reveal significant systemic changes following single-dose Ra223 and/or aPD1 therapy (Fig. S7).

Figure 4. Changes in tumor infiltration by CD8+ T-cells following Ra-223 and anti-PD1 antibody (aPD1) in TRAMPC1 mPC model.

Figure 4.

Open in a new tab

(A) Immunofluorescence for CD8+ identified cytotoxic T-cell infiltration 6 days after initiation of treatment (yellow lines are the borders of tumor tissue). (B) Intratumoral infiltration by CD8+ T-cells increased after Ra223 and increased further after combination therapy. **p<0.01; ***p<0.001, ****p<0.0001; n=7 per group.

DISCUSSION

These prospective clinical and preclinical studies together provide insights about the physiologic, mostly immune-related, effects of Ra223 in bony mPC. The clinical study aimed to examine systemic markers potentially associated with outcomes in patients receiving standard Ra223. The preclinical study aimed to correlate circulating markers with changes in the local tumor microenvironment and explore the potential of Ra223 to overcome resistance to anti-PD1 immunotherapy.

The inflammatory cytokine IL-6 emerged as candidate biomarker, consistent with previous correlative findings for IL-6 as a marker treatment resistance (20,21). IL-6 is increased in the blood after EBRT (22–25) and might be a mediator of tumor radioresistance (25). In this mPC cohort receiving systemic Ra223, a baseline plasma IL-6 above the median was associated with significantly shorter survival. Furthermore, in these patients, IL-6 trended to increase more during therapy. Given multiple known functions of IL-6, further study is needed before making mechanistic conclusions.

The angiogenic biomarker PlGF has been previously associated with tumor radioresistance through paracrine vasculature-protective effects (26) and increases in the blood following EBRT (27). Here, we found that pretreatment PlGF plasma concentration to certain degrees negatively correlated with survival of patients in this cohort according to all of the methods of statistical analysis used. When analyzing only the patients with completed all six planned Ra223 infusions, higher baseline PlGF level significantly correlated with shorter survival, and increased further during treatment in those patients. Circulating PlGF appears to be an interesting biomarker but clearly requires further study.

Cellular biomarker studies revealed several potentially immunosuppressive effects during therapy: There were rapid and stable decreases in the counts of total WBCs, lymphocytes, and neutrophils, while monocyte concentration remained unchanged. We also observed MDSC population shifts with a rise in monocytic MDSCs and a drop in granulocytic MDSCs, both predominantly in patients with shorter survival after Ra223. The mechanism and consequence of these divergent effects of Ra223 on these subpopulations should be determined in future studies.

Our clinical study did not include research biopsies. To examine the immune effects of Ra223 with or without anti-PD1 therapy in the mPC tissues, we used orthotopic preclinical models using cells (PtenSmad4-null & TRAMP-C1) derived from genetically engineered mice. In these bony mPC models, combined therapy showed modest while statistically significant efficacy. However, because of the rapid, aggressive growth of these tumors, we could not evaluate the longer-term effects on memory responses or test staggered scheduling.

The most impressive effects were observed in the TRAMP-C1 mPC model where the combination of Ra223 and ICI showed synergy (50% increase in OS) while these treatments individually were ineffective. Antitumor effects were associated with more mature tumor vessels, decreased hypoxia, and increased tumor infiltration by CD8+ T-cells. Importantly, the beneficial effects of Ra223/aPD1 therapy extended to reducing secondary metastatic burden to the lungs. Notably, simultaneous analysis of blood markers revealed no substantial immunosuppression after single-dose Ra223. These results indicate that achieving benefits when using Ra223 and anti-PD1 therapy, which were not apparent when using prolonged Ra223 treatment (28), may require mechanistic studies of both blood biomarkers and mPC tissues to characterize Ra223-induced immunomodulation.

Our preclinical data suggest that the combination of a single dose of Ra223 with anti-PD1 therapy could overcome treatment resistance by increasing CD8+ T cell infiltration throughout the tumor and delaying its aggressive growth. Given the bony localization of Ra223 and the short range of alpha-particles, it is conceivable that its effects in combination with ICI were partially due to a “neighborhood” abscopal immune-dependent effect of localized mPC irradiation, as has been demonstrated in other models with partially irradiated tumors (29,30). Also, a potential “long-distant” abscopal effect has been reported in two patients with castration-resistant mPC after Ra223 alone (31).

Along with increased CD+ T cell infiltration, we observed other traits of tumor microenvironment normalization in the TRAMP-C1 mPC model following Ra223 combined with aPD1. For example, we observed increased vessel maturity and improved tumor tissue oxygenation. These functional changes, most importantly less hypoxia, are associated with increased tumor sensitivity to low-LET radiation as can be delivered with external beam radiation therapy.

Our clinical study did not directly test Ra223/immunotherapy combinations but has direct implications for this approach, preliminarily studied by others with mixed results. Ra223 with sipuleucel-T appeared to increase the rate of >50% PSA decline and improve PFS and OS in a 32-patient study (28). But in a 45-patient study of the anti-PD-L1 antibody atezolizumab with Ra223, the response rate was just 6.8% (32). Another 45-patient study randomized participants to Ra223 with or without the anti-PD1 antibody pembrolizumab (33). They found no differences in CD4+ and CD8+ T-cell numbers in the marrow after 8 weeks on either arm, and the addition of ICI did not prolong PFS or OS.

Our clinical study has limitations. It was a single-institution study. Later timepoints for participants receiving Ra223 included fewer patients and were enriched for patients doing well on therapy as patients who progressed rapidly at the time of first on-study restaging were less likely to receive planned doses 4 through 6. The absence of a control group limits the ability to interpret the predictive versus the prognostic value of the baseline and on-treatment biomarker correlations with outcomes. Marrow sampling was not a part of this study.

The 2022 FDA approval of 177Lu-PSMA-617 for mPC and alpha-emitting PSMA-based radiopharmaceuticals in the trial pipeline highlights the need for additional insights about the effects of radiopharmaceuticals on the tumor microenvironment, particularly given that lymphopenia and leukopenia are common with 177Lu-PSMA-617 study (34). There are also multiple ongoing clinical studies examining radiopharmaceuticals in combination with immunotherapies such as pembrolizumab. Thus, there is a clear need for mechanistic insights related to systemic immune effects and potential tissue-level immunomodulation.

In summary, the inflammatory cytokine IL-6 and the angiogenic growth factor PlGF at baseline were promising circulating outcome biomarkers after Ra223 therapy in mPC. Treatment was associated with a mild decrease in some peripheral immune cell populations and a shift in the proportion of MDSCs from granulocytic to myeloid. In mouse models employed to explore the tumor microenvironment, Ra223 induced intratumoral CD8+ T-cell infiltration and proliferation. Our preclinical findings suggest that a judicious combination of Ra223 with ICI may help overcome resistance to these systemic therapies by modifying the microenvironment of bony mPC, including potentially by reducing hypoxia. Further studies of radiopharmaceuticals with ICIs are needed to gain additional mechanistic insights and facilitate the potential clinical translation of this strategy.

Supplementary Material

1
2

HIGHLIGHTS.

  • Prostate cancer is associated with systemic and local immunosuppression.

  • The radiopharmaceutical Ra223 may be immunomodulatory.

  • The inflammatory cytokine IL-6 and the angiogenic biomarker PlGF at baseline were promising prognostic markers with the clinical use of Ra223.

  • In mice, Ra223 was immunomodulatory and synergistic with immunotherapy.

  • In mice, Ra223 increased tumor CD8+ T cell infiltration and proliferation.

Sources of support:

We are grateful to our patient participants and their families. We acknowledge funding support through sponsored research agreements from Bayer for the clinical and preclinical studies (to PJS and DGD). DGD’s research is supported through NIH grants R01CA254351, R01CA260857, R01CA247441, 1P01CA261669-01, and R03CA256764, and Department of Defense grants PRCRP W81XWH-19-1-0284 and PRCRP W81XWH-21-1-0738.

Declaration of interests disclosures:

Saylor: Sponsored research agreement (institutional funds) for the clinical study described in this manuscript.

Kozin: None.

Matsui: None.

Goldberg: None.

Aoki: None.

Shigeta: None.

Mamessier: None.

Smith: Institutional funds and personal consulting fees and advisory board participation, each with/from Bayer.

Michaelson: None.

Lee: Institutional research funding from Janssen. Personal payments for advisory board consultation from Bayer, Dendreon, Janssen, Exelixis, GE, and Blue Earth.

Duda: Consultant fees from Innocoll and institutional research grants from Bayer, Exelixis, BMS and Surface Oncology.

Conflict of interest disclosure:

D.G.D. received consultant fees from Innocoll and research grants from Bayer, Exelixis, BMS, and Surface Oncology.

Footnotes

CRediT author statement:

Saylor: Conceptualization, methodology, writing – original draft preparation, Writing – review & editing, investigation, project administration, funding acquisition

Kozin: Conceptualization, methodology, writing – original draft preparation, Writing – review & editing, investigation

Matsui: Writing – review & editing, investigation

Goldberg: Methodology, data curation, Writing – review & editing

Aoki: Writing – review & editing, investigation

Shigeta: Writing – review & editing, investigation

Mamessier: Writing – review & editing, investigation

Smith: Conceptualization, Writing – review & editing, investigation

Michaelson: Writing – review & editing, investigation

Lee: Writing – review & editing, investigation

Duda: Conceptualization, methodology, writing – original draft preparation, investigation, project administration, supervision, funding acquisition

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REFERENCES:

  • 1.Parker C, Nilsson S, Heinrich D, Helle SI, O’Sullivan JM, Fossa SD, et al. Alpha emitter radium-223 and survival in metastatic prostate cancer. N Engl J Med 2013;369(3):213–23. [DOI] [PubMed] [Google Scholar]
  • 2.Abi-Ghanem AS, McGrath MA, Jacene HA. Radionuclide therapy for osseous metastases in prostate cancer. Seminars in nuclear medicine 2015;45(1):66–80 doi 10.1053/j.semnuclmed.2014.07.006. [DOI] [PubMed] [Google Scholar]
  • 3.Sartor O, Coleman RE, Nilsson S, Heinrich D, Helle SI, O’Sullivan JM, et al. An exploratory analysis of alkaline phosphatase, lactate dehydrogenase, and prostate-specific antigen dynamics in the phase 3 ALSYMPCA trial with radium-223. Ann Oncol 2017;28(5):1090–7 doi 10.1093/annonc/mdx044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Humm JL, Sartor O, Parker C, Bruland OS, Macklis R. Radium-223 in the treatment of osteoblastic metastases: a critical clinical review. Int J Radiat Oncol Biol Phys 2015;91(5):898–906 doi 10.1016/j.ijrobp.2014.12.061. [DOI] [PubMed] [Google Scholar]
  • 5.Abou DS, Ulmert D, Doucet M, Hobbs RF, Riddle RC, Thorek DL. Whole-Body and Microenvironmental Localization of Radium-223 in Naive and Mouse Models of Prostate Cancer Metastasis. J Natl Cancer Inst 2016;108(5) doi 10.1093/jnci/djv380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Turner PG, O’Sullivan JM. (223)Ra and other bone-targeting radiopharmaceuticals-the translation of radiation biology into clinical practice. The British journal of radiology 2015;88(1050):20140752 doi 10.1259/bjr.20140752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Suominen MI, Fagerlund KM, Rissanen JP, Konkol YM, Morko JP, Peng Z, et al. Radium-223 Inhibits Osseous Prostate Cancer Growth by Dual Targeting of Cancer Cells and Bone Microenvironment in Mouse Models. Clin Cancer Res 2017;23(15):4335–46 doi 10.1158/1078-0432.CCR-16-2955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Keegan A, Ricciuti B, Garden P, Cohen L, Nishihara R, Adeni A, et al. Plasma IL-6 changes correlate to PD-1 inhibitor responses in NSCLC. J Immunother Cancer 2020;8(2) doi 10.1136/jitc-2020-000678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Tsukamoto H, Fujieda K, Senju S, Ikeda T, Oshiumi H, Nishimura Y. Immune-suppressive effects of interleukin-6 on T-cell-mediated anti-tumor immunity. Cancer Sci 2018;109(3):523–30 doi 10.1111/cas.13433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Saylor PJ, Kozak KR, Smith MR, Ancukiewicz MA, Efstathiou JA, Zietman AL, et al. Changes in biomarkers of inflammation and angiogenesis during androgen deprivation therapy for prostate cancer. Oncologist 2012;17(2):212–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Beer TM, Kwon ED, Drake CG, Fizazi K, Logothetis C, Gravis G, et al. Randomized, Double-Blind, Phase III Trial of Ipilimumab Versus Placebo in Asymptomatic or Minimally Symptomatic Patients With Metastatic Chemotherapy-Naive Castration-Resistant Prostate Cancer. J Clin Oncol 2017;35(1):40–7. [DOI] [PubMed] [Google Scholar]
  • 12.Sharma P, Pachynski RK, Narayan V, Flechon A, Gravis G, Galsky MD, et al. Nivolumab Plus Ipilimumab for Metastatic Castration-Resistant Prostate Cancer: Preliminary Analysis of Patients in the CheckMate 650 Trial. Cancer cell 2020;38(4):489–99 e3 doi 10.1016/j.ccell.2020.08.007. [DOI] [PubMed] [Google Scholar]
  • 13.Antonarakis ES, Piulats JM, Gross-Goupil M, Goh J, Ojamaa K, Hoimes CJ, et al. Pembrolizumab for Treatment-Refractory Metastatic Castration-Resistant Prostate Cancer: Multicohort, Open-Label Phase II KEYNOTE-199 Study. J Clin Oncol 2020;38(5):395–405 doi 10.1200/JCO.19.01638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Kwon ED, Drake CG, Scher HI, Fizazi K, Bossi A, van den Eertwegh AJ, et al. Ipilimumab versus placebo after radiotherapy in patients with metastatic castration-resistant prostate cancer that had progressed after docetaxel chemotherapy (CA184–043): a multicentre, randomised, double-blind, phase 3 trial. Lancet Oncol 2014;15(7):700–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Hansen AR, Massard C, Ott PA, Haas NB, Lopez JS, Ejadi S, et al. Pembrolizumab for advanced prostate adenocarcinoma: findings of the KEYNOTE-028 study. Ann Oncol 2018;29(8):180713 doi 10.1093/annonc/mdy232. [DOI] [PubMed] [Google Scholar]
  • 16.Donlon NE, Power R, Hayes C, Reynolds JV, Lysaght J. Radiotherapy, immunotherapy, and the tumour microenvironment: Turning an immunosuppressive milieu into a therapeutic opportunity. Cancer letters 2021;502:84–96 doi 10.1016/j.canlet.2020.12.045. [DOI] [PubMed] [Google Scholar]
  • 17.Weichselbaum RR, Liang H, Deng L, Fu YX. Radiotherapy and immunotherapy: a beneficial liaison? Nat Rev Clin Oncol 2017;14(6):365–79 doi 10.1038/nrclinonc.2016.211. [DOI] [PubMed] [Google Scholar]
  • 18.Kim JW, Shin MS, Kang Y, Kang I, Petrylak DP. Immune Analysis of Radium-223 in Patients With Metastatic Prostate Cancer. Clin Genitourin Cancer 2018;16(2):e469–e76 doi 10.1016/j.clgc.2017.10.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Goyal L, Zheng H, Abrams TA, Miksad R, Bullock AJ, Allen JN, et al. A Phase II and Biomarker Study of Sorafenib Combined with Modified FOLFOX in Patients with Advanced Hepatocellular Carcinoma. Clin Cancer Res 2019;25(1):80–9 doi 10.1158/1078-0432.CCR-18-0847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Hodge DR, Hurt EM, Farrar WL. The role of IL-6 and STAT3 in inflammation and cancer. Eur J Cancer 2005;41(16):2502–12 doi 10.1016/j.ejca.2005.08.016. [DOI] [PubMed] [Google Scholar]
  • 21.Schroeder A, Herrmann A, Cherryholmes G, Kowolik C, Buettner R, Pal S, et al. Loss of androgen receptor expression promotes a stem-like cell phenotype in prostate cancer through STAT3 signaling. Cancer Res 2014;74(4):1227–37 doi 10.1158/0008-5472.CAN-13-0594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Sepah SC, Bower JE. Positive affect and inflammation during radiation treatment for breast and prostate cancer. Brain Behav Immun 2009;23(8):1068–72 doi 10.1016/j.bbi.2009.06.149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Christensen E, Pintilie M, Evans KR, Lenarduzzi M, Menard C, Catton CN, et al. Longitudinal cytokine expression during IMRT for prostate cancer and acute treatment toxicity. Clin Cancer Res 2009;15(17):5576–83. [DOI] [PubMed] [Google Scholar]
  • 24.Lopes CO, Callera F. Three-dimensional conformal radiotherapy in prostate cancer patients: rise in interleukin 6 (IL-6) but not IL-2, IL-4, IL-5, tumor necrosis factor-alpha, MIP-1-alpha, and LIF levels. Int J Radiat Oncol Biol Phys 2012;82(4):1385–8 doi 10.1016/j.ijrobp.2011.04.040. [DOI] [PubMed] [Google Scholar]
  • 25.Wu CT, Chen MF, Chen WC, Hsieh CC. The role of IL-6 in the radiation response of prostate cancer. Radiat Oncol 2013;8:159 doi 10.1186/1748-717X-8-159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Kazimova T, Tschanz F, Sharma A, Telarovic I, Wachtel M, Pedot G, et al. Paracrine placental growth factor signaling in response to ionizing radiation is p53-dependent and contributes to radioresistance. Mol Cancer Res 2021. doi 10.1158/1541-7786.MCR-20-0403. [DOI] [PubMed]
  • 27.Gkika E, Adebahr S, Brenner A, Schimek-Jasch T, Radicioni G, Exner JP, et al. Changes in Blood Biomarkers of Angiogenesis and Immune Modulation after Radiation Therapy and Their Association with Outcomes in Thoracic Malignancies. Cancers (Basel) 2021;13(22) doi 10.3390/cancers13225725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Marshall CH, Fu W, Wang H, Park JC, DeWeese TL, Tran PT, et al. Randomized Phase II Trial of Sipuleucel-T with or without Radium-223 in Men with Bone-metastatic Castration-resistant Prostate Cancer. Clin Cancer Res 2021;27(6):1623–30 doi 10.1158/10780432.CCR-20-4476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Markovsky E, Budhu S, Samstein RM, Li H, Russell J, Zhang Z, et al. An Antitumor Immune Response Is Evoked by Partial-Volume Single-Dose Radiation in 2 Murine Models. Int J Radiat Oncol Biol Phys 2019;103(3):697–708 doi 10.1016/j.ijrobp.2018.10.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Billena C, Khan AJ. A Current Review of Spatial Fractionation: Back to the Future? Int J Radiat Oncol Biol Phys 2019;104(1):177–87 doi 10.1016/j.ijrobp.2019.01.073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Kwee SA, Lim J, Coel MN. Soft Tissue Response on 18F-Fluorocholine PET/CT in Metastatic Castrate-Resistant Prostate Cancer Treated With 223Ra-Dichloride: A Possible Abscopal Effect? Clin Nucl Med 2017;42(11):868–71 doi 10.1097/RLU.0000000000001807. [DOI] [PubMed] [Google Scholar]
  • 32.Fong L, Morris MJ, Sartor O, Higano CS, Pagliaro L, Alva A, et al. A Phase Ib Study of Atezolizumab with Radium-223 Dichloride in Men with Metastatic Castration-Resistant Prostate Cancer. Clin Cancer Res 2021;27(17):4746–56 doi 10.1158/1078-0432.CCR-21-0063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Choudhury AD, Kwak L, Cheung A, Tripathi A, Pace AF, Van Allen EM, et al. Randomized phase II study evaluating the addition of pembrolizumab to radium-223 in metastatic castration-resistant prostate cancer. J Clin Oncol 2021;39 doi 10.1200/JCO.2021.39.6_suppl.98. [DOI] [PMC free article] [PubMed]
  • 34.Sartor O, de Bono J, Chi KN, Fizazi K, Herrmann K, Rahbar K, et al. Lutetium-177-PSMA-617 for Metastatic Castration-Resistant Prostate Cancer. N Engl J Med 2021. doi 10.1056/NEJMoa2107322. [DOI] [PMC free article] [PubMed]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1961949-supplement-1.docx (2MB, docx)
2
NIHMS1961949-supplement-2.docx (2.2MB, docx)

RESOURCES