WHAT IS ALREADY KNOWN ON THIS TOPIC

Androgen deprivation can work synergistically with external beam radiation therapy to prolong time to progression and survival of patients with high-risk localized prostate cancer. The combination of androgen deprivation and systemic targeted radionuclide therapy, however, and the optimal sequence of this combination, has not been previously evaluated.

WHAT THIS STUDY ADDS

In murine models of prostate cancer, we demonstrate that there is a sequence preference to the delivery of androgen deprivation and targeted radionuclide therapy, and this is mediated by differences in T cells and myeloid cells within the tumor immune microenvironment. The antitumor efficacy of this combination was improved by the addition of agents that depleted or reduced the migration of Gr-1+myeloid-derived suppressor cells.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYBackground

Radiation therapy (RT) has been one of the mainstay treatments for prostate cancer. External beam RT (EBRT) can be curative for localized prostate cancer but has traditionally been limited to palliation for widely metastatic disease due to the inability to radiate all sites of metastasis.1 Systemic administration of radionuclides that are preferentially taken up in bone has been used to treat painful bone metastases. These radionuclides include beta-emitting 89Sr and 153Sm, and US Food and Drug Administration (FDA)-approved alpha-emitting 223RaCl2 (Xofigo) for the treatment of metastatic castration resistant prostate cancer (mCRPC) with bone metastases.2–4 This approach, using targeted radionuclides to treat all metastatic diseases simultaneously, with relative sparing of healthy tissue, is called radiopharmaceutical therapy or targeted radionuclide therapy (TRT).

While these TRT agents have been useful for patients with disease metastasized exclusively to the bone, they are not effective for those with other disease sites. Hence, other investigations have focused on compounds that specifically target cancer cells rather than the bone. One of the most studied targets for prostate tumor-directed radiation delivery is prostate-specific membrane antigen (PSMA) which is highly expressed in prostate cancer cells. Early attempts used 177Lu or 90Y conjugated to an antibody specific for PSMA (J591), which was well tolerated and promising in early clinical trials.5 6 Further efforts focused on the development of small molecules such as [18F]DCFPyL and PSMA-11, which have both been used in positron emission tomography (PET)/CT diagnostic imaging.7 8 Another PSMA analog, PSMA-617, has also been labeled with radionuclides suitable for therapy (eg, 177Lu, 225Ac) of recurrent prostate cancer.9 177Lu-PSMA-617 was the first cancer-targeted TRT agent that received FDA approval for the treatment of mCRPC on the basis of it demonstrating a survival benefit compared with standard of care androgen receptor-targeted therapy, although by only 4 months.10

While androgen deprivation therapy (ADT) and RT are standard treatments for localized prostate cancer, there has been relatively limited exploration of ADT combined specifically with TRT.11 Apart from their independent cytotoxic effects, there is evidence to suggest that ADT synergistically works with RT by preventing DNA repair.12 13 However, the order in which ADT and TRT are best administered has not been rigorously studied.14 Recent data indicate that RT and ADT can distinctly influence the tumor immune microenvironment.15 ADT enhances vulnerability to CD8+T cell-mediated destruction, triggers thymus regeneration, amplifies naive T cell production, augments immune cell infiltration from myeloid and lymphocyte populations, and elevates antibody responses against prostate-specific antigens.16–20 However, ADT also triggers a significant secretion of IL-8 in human prostate tumors, which can lead to the accumulation of intratumoral myeloid-derived suppressor cells (MDSCs), which may impede T-cell activity.21 Conversely, RT elicits inflammatory responses, including the upregulation of MHC-I expression on tumor cells, enhancement of antigen cross-presentation by antigen-presenting cells, activation of the Fas/Fas ligand (Fas-L) signaling pathway, targeting of immune-suppressive populations like regulatory T cells (Tregs), and the induction of immunogenic cell death.22 23 In combination, ADT and RT can synergistically enhance tumor immunity, modulating both local and systemic antitumor immune responses.24 Therefore, investigating effective strategies for their combination, including considerations such as the timing and sequence of ADT with RT, as well as the integration of newer systemic TRT agents, is crucial.

Our group has employed alkylphosphocholines (APCs) as TRT agents given that they can specifically accumulate within tumor cells by integrating into lipid rafts.25 First-generation 131I-NM404 is currently under investigation as a potential monotherapy treatment for metastatic multiple myeloma and other cancer types.26–28 We have recently focused on the assessment of a second-generation APC chelate, called NM600, which can be tagged with different radiometals. By employing the radiometal 86Y, one can visualize tumors and perform dosimetry measurements by PET/CT imaging.29 Alternatively, through labeling with the isotopic pair, 90Y, one can administer therapeutic radiation.30 31 This innovative approach, using Y-NM600 for both imaging and therapy, has demonstrated success in numerous preclinical models.29 30 32 However, its applicability to prostate cancer used in conjunction with ADT has not been previously investigated.

In this report, we explored the combination of TRT using 90Y-NM600 with ADT in murine prostate models and specifically examined the effects of this combination on the tumor immune microenvironment. Our findings revealed that the effectiveness of this combination was influenced by the order of administration. ADT followed by TRT (ADT→TRT) showed superior enhancement of antitumor responses compared with the reverse sequence of TRT followed by ADT (TRT→ADT). We demonstrated that this disparity was due, in part, to the presence of infiltrating MDSCs, which impaired the function of CD8+T cells. Furthermore, we showed that the efficacy of antitumor responses could be improved by inhibiting the migration of MDSCs in vivo using a CXCR2 antagonist. These findings underscore the significance of understanding the mechanisms through which ADT and TRT influence the tumor microenvironment, enabling the optimal timing and choice of combination therapies for prostate cancer.

Materials and methodsRadiosynthesis of 90Y-NM600Cell lines

TRAMP-C1 (CRL-2730) and Myc-CaP (CRL-3255) cell lines were obtained from ATCC (Manassas, Virginia, USA), maintained according to ATCC recommendations, and tested for mycoplasma contamination.

Mice

FVB/NJ mice (stock #001800) and C57BL/6J mice (stock #000664) were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA) and housed in micro insulator cages under aseptic conditions. NRG mice were graciously provided by Dr. Paul Sondel (University of Wisconsin-Madison). All animal studies were conducted under an IACUC-approved protocol.

Tumor implantation and tumor growth studies

1×106 Myc-CaP cells, resuspended in PBS, were implanted subcutaneously into the right flank of male FVB mice aged 4–6 weeks old or male NRG mice aged 6–10 weeks old. Similarly, wild-type male C57BL/6J mice aged 4–6 weeks were injected subcutaneously with 1×106 TRAMP-C1 cells in 1:1 ratio in phosphate buffered saline (PBS): Matrigel (Corning, NY. CB354248) into the right flank. 12–15 days postinjection, when tumors were palpable and similarly sized (0.2–0.3 cm3), mice were randomized into treatment groups. Tumors were measured twice weekly via calipers until the tumors reached 2 cm3. Tumor volumes were calculated as (long axis×short axis2)/2.

Androgen deprivation therapy

Mice were treated subcutaneously with either degarelix (25 mg/kg) or a vehicle sham treatment (PBS) every 28 days starting when the tumor volume reached ~0.2–0.3 cm3 in size.

Radiosynthesis of 86/90Y-NM600

Briefly, 86YCl3 was provided by the University of Wisconsin-Madison cyclotron group after proton bombardment of enriched [86Sr] SrCO3 solid targets in a PETtrace biomedical cyclotron and elution of 86Y from a diglycolamide extraction resin column.33 Clinical grade 90YCl3 and NM600 were obtained from PerkinElmer (Shelton, CT) and Archeus Technologies (Madison, WI) respectively. NM600 was radiolabeled with 90Y or 86Y and purified as previously described.29 30 Briefly, 185–370 MBq (5–10 mCi) of 86/90Y was buffered with 0.1 M NaOAc (pH 5.5) and mixed with 55–110 nmol (50–100 µg). The reaction was incubated for 30 min at 95°C under constant shaking (500 rpm). 86/90Y-NM600 was purified by a solid-phase extraction cartridge (HLB; Waters) and eluted in 2 mL of 200-proof ethanol. The eluate was then evaporated and dried under a nitrogen stream, and 86/90Y-NM600 was reconstituted in excipient (saline containing 0.1% v/v Tween20). Radiochemical yield was assessed by instant thin-layer chromatography (iTLC) using silica-impregnated paper as the stationary phase and run using 50 mM ethylenediaminetetraacetic acid, which moves the free radiometals with the solvent front (Rf=1) while 86/90Y-NM600 remains at the origin (Rf=0). iTLC chromatograms were developed using a cyclone phosphor-plate imager and analyzed with Optiquant software (PerkinElmer). Radiochemical purity and stability were determined via radiolabeled high-performance liquid chromatography (HPLC) using a reverse-phase 250×3.00 mm C18 Luna 5 µm 100 Å column (Phenomenex) and a water:acetonitrile gradient (5% MeCN: 0–2 min; 5%–65% MeCN: 2–30 min; 65%–90% MeCN: 30–35 min; 90%–5% MeCN: 35–45 min). The final radiochemical purity obtained consistently surpassed 95% with an average molar specific activity of 18 GBq/µmol for both 90Y-NM600 and 86Y-NM600 (n>5). Additionally, HPLC chromatograms indicated that both 90Y-NM600 and 86Y-NM600 were stable in mouse serum over at least 48 hours.29

Dosimetry estimation

Dosimetry estimations were performed as previously reported using a Monte Carlo-based dosimetry assessment platform, Radionuclide Assessment Platform for Internal Dosimetry.34 35 The dosimetry and biodistribution of 90Y-NM600 have been previously published for murine Myc-CaP and TRAMP-C1 prostate tumors.29 32

TRT administration

90Y-NM600 250µCi~9.25 MBq was injected into the tail vein of tumor-bearing mice 1 week before or after the start of ADT. Based on dosimetry studies, a single dose of 250 µCi injected activity delivered 5–6 Gy absorbed dose to TRAMP-C1 tumors and 16–20 Gy to Myc-CaP tumors.29 32

Antibody treatments

All antibody treatments, anti-CD4 (BioXcell BP0003-1), anti-CD8 (BioXcell BP0061) and IgG2a isotype (BioXcell BP0085), were administered as 200 µg intraperitoneal injections, on days 2, 4, and 6 post-ADT or TRT. 200 µg anti-mouse Gr-1 antibody (clone RB6-8C5) (BD Pharmingen 552985) was administered intraperitoneally three times a week post-TRT administration.

Flow cytometry

Tumors were collected at different time points, then digested for 1–2 hours at 37°C in mouse cell culture medium: RPMI 1640 with L-glutamine, 10% fetal calf serum, 200 U/mL Pen/Strep, 5% sodium pyruvate, 5% HEPES, and 50 µM β-MeOH supplemented with 2 mg/mL collagenase, 0.2 mg/mL DNAse I, and 1 tablet protease inhibitor (Sigma-Aldrich, St. Louis, MO, 11697498001) per 50 mL digest solution. Digests were then passed through 100 µm screens. 5×106 cells were plated and Fc blocked (BD, Franklin Lakes, NJ, 553142) for 20 min at 4°C. Cells were then stained for 30 min at 4°C with the viability dye Ghost Dye Red 780 (Tonbo 13-0865 T100) and the following antibodies: CD11b-BB515 (BD 564454), CD25-BB700 (BD 566498), GR-1-PE-CF594 (BD 562710), CD3-PE-Cy7 (eBiosciences Thermo Fisher Scientific, Waltham, MA 25-0031-82), MHCII-BV421 (Biolegend San Diego, CA 107632), CD45-BV510 (BD 563891), CD4-BV605 (Biolegend 100451), CD19-BV711 (BD 563157), CD11c-APC (BD 550261), CD8-AF700 (100730), CD44-AF488 (Biolegend 103016), CD45- PerCP-Cy5.5 (Biolegend 103132), KLRG-1-PE (Biolegend 138408), CD69-PE-CF594 (BD 562455), CD62L-BV510 (Biolegend 104441), CD103-BV605 (Biolegend 121453), CD27-BV785 (Biolegend 124241), CD4-APC-Cy7 (Biolegend 561830). Cells were then fixed and permeabilized with the eBiosciences Foxp3/Transcription Factor Staining Buffer Set overnight at 4°C (Thermo Fisher 00-5523-00). Cells were then stained with intracellular antibodies for 30 min at 4°C: FoxP3-PE (Thermo Fisher 12-5773-82), Ki67-BV421 (BD 562899). Flow cytometry was performed on a Thermo Fisher Attune NxT cytometer and data were analyzed using FlowJo V.10. Gates were set according to a fluorescence-minus-one control. Flow cytometry data were reported as either the percentage of populations among all CD45+ events or as a frequency per gm of tumor tissue.

CXCR2 antagonist

CXCR2 antagonist, reparixin (Selleckchem, Houston, Texas), was reconstituted in Tween-80 and PBS in a 1:4 ratio and administered subcutaneously at 5 mg/kg on the left flank thrice a week for 3 weeks post-TRT administration.

In vitro studiesCD8 T cell suppression assay

Spleens were harvested from naïve FVB mice and passed through 100 µm screens. CD8+T cells were isolated from splenocytes via immunomagnetic negative selection (StemCell #19853), and then labeled with carboxyfluorescein succinimidyl ester (Biolegend #423801) according to the manufacturer’s instructions. Tumors were collected from treated tumor-bearing mice on day 36, processed into single-cell suspensions as above, and CD11b+Gr-1+Ly-6G+MDSCs were isolated (Miltenyi Biotec #130-094-538). 1×105 labeled CD8+T cells were cultured together with MDSCs at a 1:1 ratio. CD8+T cells were stimulated with anti-CD3/anti-CD28 coated beads (Thermo Fisher 11 456D) at a ratio of 2 beads per CD8+T cell. Cells were cultured with 30 units/mL of human IL-2 for 72 hours in 96-well plates before analysis via flow cytometry.

ELISA

ELISA was performed as previously described.36 Briefly, Immulon plates (Thermo Fisher, Waltham, Massachusetts, USA) were coated with anti-mouse IFNγ antibody (BD #551216) and incubated overnight at 4°C. Plate were then blocked with PBS/1% BSA before adding standards (BD #554587) or cell culture supernatants and incubated overnight at 4°C. The next day, a biotin-conjugated anti-mouse IFNγ antibody was added (BD #554410), followed by avidin-HRP (BioRad Hercules, CA, 170-6528). TMB Substrate (Kirkegaard and Perry, Gaithersburg, MD, 50-76-01) was added and OD was measured at 450 nm.

Luminex assay

50 µL of sera or conditioned media from in vitro assays was evaluated for 26 different cytokines and chemokines using the Cytokine & Chemokine 26-Plex Mouse ProcartaPlex Panel 1 (Thermo Fisher EPX260-26088-901) according to the manufacturer’s instructions. The plate was read on a Luminex MagPix instrument. Analytes were divided according to their type, Th1 (IFN gamma, IL-12p70, IL-18, IL-27, IL-2, TNF alpha, GMCSF, IL-1 beta), Th2 (IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GMCSF), Th17 (IL-17A, IL-22, IL-23), and chemokines (CXCL10, CXCL1, CCL2, CCL7, CCL3, CCL4, CXCL2, CCL5, CCL11).

In vitro chemotaxis assay

3×105 cells (Myc-CaP cells and/or T cells including CD4 and CD8 T cells isolated from naïve FVB mice splenocytes) were plated in regular media, charcoal-stripped media (regular media with charcoal-stripped FBS (Thermo#12676029), 90Y containing media (23.3µCi~0.86 MBq of 90Y per 1 mL media) or 90Y-containing charcoal-stripped media, in 6-well plates (n=3 replicates per media condition). Wells containing T cells were stimulated with anti-CD3/anti-CD28 coated beads at a ratio of 2 beads per CD8+T cell. Supernatants were collected after incubation for 72 hours. 1×105 MDSCs isolated from tumors as described above were added to the top chamber of the transwell and cultured for 12 hours with the conditioned media in the bottom well. In related experiments, a 5 ng/mL recombinant CXCL1 was used as a positive control, and MDSCs were pretreated with 4 mM reparixin. In other related experiments, conditioned media from T cells were added to the Myc-CaP conditioned media in a 1:1 ratio. After incubation, cells were collected from the bottom well, stained, and analyzed via flow cytometry. The absolute number of MDSCs was determined and the percent migration was calculated as the fraction of MDSCs present in the bottom well of the total number of MDSCs plated in the transwell.

Statistical analysis

Tumor growth data, comparing group means among treatment groups, were analyzed by fitting a linear mixed-effects model with Geisser-Greenhouse correction. The data were analyzed via analysis of variance followed by Tukey’s multiple-comparison test. Survival analysis was conducted using a Mantel-Cox log-rank test. For all comparisons, p values ≤0.05 were considered statistically significant with asterisks *p<0.05, **p<0.01, and ***p<0.001. All statistical analyses were performed using GraphPad Prism software V.10.0.3.

ResultsCombination of ADT and TRT with ADT prior to TRT (ADT→ TRT) significantly improved antitumor responses in murine prostate tumor models

We studied the effects of 90Y-NM600 in combination with ADT in two separate murine prostate tumor models, Myc-CaP and TRAMP-C1. As depicted in figure 1A, MyC-CaP tumor cells were implanted subcutaneously in male FVB mice, and when tumors reached a volume of 0.2–0.3 cm3 they were treated with degarelix. TRT (250µCi~9.25 MBq of 90Y- NM600, delivering~16 Gy) was given 1 week before or after degarelix. When ADT was delivered prior to TRT (ADT→TRT), there was a significant tumor growth delay (figure 1B and online supplemental figure 1A) and improved overall survival (figure 1C). Because ADT and TRT can have different effects depending on the day they are administered relative to tumor volume, in a second study, ADT was again used before or after TRT, but this time fixing the day on which TRT was administered (figure 1D). As before, the ADT→TRT combination significantly delayed tumor growth (figure 1E and online supplemental figure 1B and 2) and improved overall survival (figure 1F) compared with the monotherapies or TRT→ADT combination. ADT→TRT also significantly improved antitumor responses and overall survival in a prostate tumor model in which TRAMP-C1 tumor cells were implanted in C57BL/6 mice (figure 1G–I and online supplemental figure 1C). However, there was no evidence of improved treatment response or overall survival when Myc-CaP cells were implanted in NRG mice lacking functional T cells (figure 1J–L and online supplemental figure 1 D). Overall, these findings demonstrated that ADT and TRT had a stronger antitumor effect in combination and were dependent on the order of administration, with ADT→TRT leading to superior antitumor responses, and this was likely immune cell dependent.

Figure 1

Combination of ADT and TRT with ADT prior to TRT (ADT→TRT) significantly improved antitumor responses in murine prostate tumor models. FVB mice were implanted with Myc-CaP tumor cells and treated with degarelix (ADT), with TRT delivered before or after ADT, and followed for tumor growth (n=10 per group). Shown is a schema (A), tumor growth curves (B), and Kaplan-Meier curves depicting survival (time to a tumor size of 2 cm3 or death, C). A similar study fixed the day of TRT with ADT delivered before or after (n=10 per group). Shown is the schema (D), tumor growth curves (E), and Kaplan-Meier survival curves (time to tumor size of 2 cm3 or death, F). Similarly, male C57BL/6 were implanted with TRAMP-C1 tumor cells (n=5 per group) and treated with ADT delivered before or after TRT. Shown is the schema (G), tumor growth curves (H), and Kaplan-Meier survival curves (time to tumor size of 2 cm3 or death, I). In addition, Myc-CaP tumor cells were implanted in male NRG T-cell deficient mice, treated with ADT and/or TRT as before (n=10 per group), and followed for tumor growth (schema in J). Shown are the tumor growth curves (K) and Kaplan-Meier survival curves (L). For tumor growth curves, asterisks demonstrate significant (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) differences as assessed by linear mixed effects model with Geisser-Greenhouse correction and Tukey’s multiple comparisons test with individual variances; Kaplan-Meier curves were compared using the log-rank test with asterisks indicating *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Results are from one experiment and are representative of two independent experiments for each study (online supplemental figure 2). ADT, androgen deprivation therapy; TRT, targeted radionuclide therapy.

CD4+T and CD8+T cells persisted in the tumor microenvironment in the ADT→TRT sequence whereas significant increases in MDSCs were observed in the TRT→ADT sequence

We next sought to understand the effect of sequencing these treatments on the tumor immune microenvironment. A similar study was performed as in figure 1D, but tumors were collected at several time points following treatment for evaluation of immune cell compositions via flow cytometry, as shown in figure 2A. Representative flow plots for T cells and MDSCs are shown in figure 2B (and gating strategy shown in online supplemental figure 3). We found that CD4+T and CD8+T cells persisted in the tumor microenvironment until day 32 in the ADT→TRT treated mice (figure 2C,D) compared with the TRT→ADT treated mice (figure 2E,F, and online supplemental figure 4). Increases in MDSCs were not observed in ADT→TRT mice until day 39, and there were no significant changes in regulatory CD4+T cells following treatment (figure 2G,H). Notably, MDSCs were significantly increased in the TRT→ADT group immediately after TRT treatment and this increase was further accentuated with the subsequent administration of ADT (figure 2I), whereas there were no significant changes in regulatory CD4+T cells (figure 2J). Taken together, these data suggested that the balance of CD4+T cells, CD8+T cells and MDSC affected by these treatments might have contributed to the preferred treatment sequence.

Figure 2

CD4+T and CD8+T cells persist in the tumor microenvironment in the ADT→TRT sequence combination whereas significant increases in MDSCs were observed in the TRT→ADT sequence. Myc-CaP tumor cells were implanted in male FVB mice and treated with ADT and/or TRT, with tumors sampled at different days for flow cytometry analysis (n=6 per group per time point). Shown are a schema (A) and representative dot plots (B) of CD4+CD3+ and CD8+CD3+ T cells and CD11b+Gr1+ MDSCs in ADT→TRT (left panels) and TRT→ADT groups (right panels) collected on day 32. CD4+T cells (C, E), CD8+T cells (D, F), and CD11b+Gr-1+ MDSC (G, I) are shown as a percentage of CD45+cells. CD4+FoxP3+ Treg (H, J) are shown as a percentage of CD4+cells. C–J were compared using one-way ANOVA with Tukey’s multiple comparisons test asterisks indicating *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Results are from one experiment and are representative of two independent experiments (online supplemental figure 4). ADT, androgen deprivation therapy; ANOVA, analysis of variance; MDSCs, myeloid-derived suppressor cells; TRT, targeted radionuclide therapy.

ADT→TRT led to persistence of activated and memory CD8+ T cells while these were significantly reduced in the TRT→ADT group

A similar study was performed to further characterize CD8+T cells (figure 3A) (with the gating strategy shown in online supplemental figure 5). Tumor-infiltrating CD8+T cells from mice treated in the ADT→TRT sequence were found to have increased proliferation (Ki67+) and activation (CD69) (figure 3B,C) compared with the TRT→ADT treatment sequence (figure 3D,E). Notably, memory CD8+T cells in the ADT→TRT sequence persisted, including effector and resident memory populations (figure 3F, G, J and K). Conversely, the TRT→ADT sequence led to a significant reduction in memory CD8+T cells, notably effector and resident memory populations (figure 3H, I, L and M). In summary, these findings indicate that the ADT→TRT treatment sequence facilitated the sustained presence of activated and memory CD8+T cells, whereas these populations were substantially diminished in mice initially treated with TRT.

Figure 3

ADT→TRT led to persistence of activated and memory CD8+T cells while these were significantly reduced in the TRT→ADT group. Myc-CaP tumor cells were implanted in male FVB mice, treated with ADT and/or TRT as before, and tumors were sampled at different days (n=5 per group per time point) for flow cytometry analysis of CD8+CD3+ cells (schema in A). Untreated CD8+T cells were collected from animals on day 18. Left panels indicate data from ADT→TRT treated animals, and right panels indicate data from TRT→ADT treated animals. Data indicate the number of each population per gram of tumor for Ki67+CD8+CD3+ T cells (B and D), CD69 MFI on CD8+T cells (C, E), CD44+ memory CD8+ T cells (F, H), CD44+CD27+CD62L+ central memory CD8+T cells (G and I), CD44+CD27−CD62L− effector memory CD8+T cells (J, L), and CD69+CD103+ resident memory CD8+T cells (K, M). Comparisons were made using one-way ANOVA with Tukey’s multiple comparisons test asterisks indicating *p<0.05, **p<0.01. Results are from one experiment and are representative of two independent experiments. ADT, androgen deprivation therapy; ANOVA, analysis of variance; TRT, targeted radionuclide therapy.

T cell depletion reduces the antitumor efficacy of the combination of ADT and TRT

We next sought to understand if T cells were required in mediating differences in antitumor responses by depleting these populations immediately after ADT or TRT (figure 4A). In the ADT→TRT group, depleting CD4+T or CD8+T cells resulted in a slightly accelerated tumor growth, although the difference was not statistically significant. However, in the TRT→ADT group, CD8+T cell depletion led to significantly more rapid tumor growth (figure 4B and online supplemental figure 6). Regardless of the combination sequence, depletion of T cells worsened survival (figure 4C).

Figure 4

The antitumor efficacy of the combination of ADT and TRT worsened in the absence of T cells. Myc-CaP tumor cells were implanted in male FVB mice, treated with ADT and/or TRT as before, and mice received IgG, anti-CD4 or anti-CD8 depleting antibodies between these treatments (schema in A). Shown are the tumor growth curves (B) and Kaplan-Meier survival curves (C). For tumor growth curves, comparisons were made by linear mixed effects model with Geisser-Greenhouse correction and Tukey’s multiple comparisons test with individual variance; Kaplan-Meier curves were compared using the log-rank test. Asterisks demonstrate significant differences (*p<0.05, **p<0.01, ***p<0.001). Results shown are each from one experiment (n=7 per group). ADT, androgen deprivation therapy; TRT, targeted radionuclide therapy.

MDSC depletion significantly improved antitumor responses and increased infiltration of CD4+ and CD8+ T cells into prostate tumors

We next wished to determine whether tumor infiltrating MDSCs that were present following TRT were functionally immunosuppressive. MDSCs were obtained from mice treated with TRT with or without ADT and evaluated for their effects on CD8+T cell proliferation (figure 5A). We found that MDSCs obtained from tumors of mice treated with TRT alone had a slight suppressive effect on CD8+T cell proliferation, however, MDSCs from mice subjected to the TRT→ADT treatment markedly suppressed CD8+T cell proliferation (figure 5B). MDSC from mice treated with either TRT alone or TRT→ADT similarly suppressed IFNγ secretion from CD8+T cells stimulated with anti-CD3/anti-CD28 beads (figure 5C). These data demonstrate that MDSCs infiltrating tumors in mice treated with TRT were still functionally active. We next used clodronate liposomes or anti-Gr1 antibody to deplete these myeloid populations in mice treated with TRT→ADT (figure 5D). Either of these treatments resulted in significantly greater tumor control compared with control mice (figure 5E and online supplemental figure 7). These treatments led to a significant decrease in tumor-infiltrating MDSCs (figure 5F), as well as slight increases in tumor-infiltrating CD4+ (figure 5G) and CD8+ (figure 5H) T cells.

Figure 5

Depletion of MDSCs significantly improved antitumor responses and increased infiltration of CD4+and CD8+ T cells into prostate tumors. CD11b+Gr-1+ MDSC were collected from Myc-CaP tumor-bearing mice that had been treated with TRT alone or TRT→ADT. MDSCs were cocultured with naïve, CFSE-labeled CD8+T cells and stimulated with anti-CD3/anti-CD28 beads (schema in A). After 72 hours, flow cytometry was conducted to evaluate CD8+T cell proliferation by loss of CFSE (B), and culture supernatants were evaluated for IFNγ concentration (C). Myc-CaP tumor cells were implanted in male FVB mice, treated with TRT→ADT as before, and mice received control liposomes, clodronate liposomes, (n=7 per group) or anti-Gr-1 antibody (n=3) as indicated (schema in D). Shown are the tumor growth curves (E). Tumors were collected on day 54 (n=3 per group) and evaluated by flow cytometry for CD11b+Gr-1+ MDSC (F), CD4+T cells (G), and CD8+T cells (H). For tumor growth curves, asterisks demonstrate significant (p<0.05) differences as assessed by linear mixed effects model with Geisser-Greenhouse correction. For F–H, comparisons were made using one-way ANOVA with Tukey’s multiple comparisons test; asterisks indicate *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Results are from one experiment, and representative of results from an independent experiment (online supplemental figure 7B). ADT, androgen deprivation therapy; ANOVA, analysis of variance; CFSE, carboxyfluorescein succinimidyl ester; MDSCs, myeloid-derived suppressor cells; TRT, targeted radionuclide therapy.

Cytokines and chemokines secreted by tumor cells promote MDSC infiltration into tumors

We next explored the potential mechanism of tumor infiltration by MDSCs by investigating the effects of the combination treatments on the cytokines and chemokines present in the sera following these different treatments (figure 6A and online supplemental figure 8). As shown in figure 6B–F, CXCL1, CXCL2 and CCL5, all chemokines associated with myeloid cell migration, were significantly increased in sera of mice treated with the TRT→ADT sequence relative to the ADT→TRT sequence. To determine which cell types may be involved in MDSC recruitment, a chemotaxis assay was performed using tumor cells, T cells, or the combination, in a testosterone-replete or testosterone-deficient medium (figure 6G). As shown in figure 6H, tumor cells primarily contributed to MDSC migration. The presence of T cells slightly reduced the migration of MDSC. Similar differences were observed using testosterone replete or testosterone-deficient medium media containing 90Y (online supplemental figure 9A). CXCL1 and CXCL2 were increased significantly in Myc-CaP conditioned media (figure 6I,J) while CCL2, CCL3, CCL5 were increased in conditioned media containing Myc-CaP and T cells (figure 6K–M). No significant changes were observed in other chemokines and cytokines (online supplemental figures 9B and 10).

Figure 6

Cytokines and chemokines secreted by tumor cells promote MDSC infiltration into tumors. Myc-CaP tumor cells were implanted in male FVB mice, treated with ADT and/or TRT as before, and sera were collected at different days (n=2 per group per time point) for cytokine and chemokine quantification (schema in A). Shown are concentrations of cytokines in pg/mL for CXCL1 (B), CXCL2 (C), CCL2 (D), CCL3 (E), and CCL5 (F). Culture supernatant from cultured Myc-CaP cells, naïve CD4+ and CD8+ T cells, the combination, or media alone (containing testosterone-replete or testosterone-deficient serum) were placed in the bottom of transwell chambers, with MDSC collected from treated mice placed in the upper chambers (schema in G). The percentage of MDSC that migrated to the bottom chamber was determined by flow cytometry (H), and the conditioned media were evaluated for CXCL1 (I), CXCL2 (J), CCL2 (K), CCL3 (L), and CCL5 (M). B–F and H–M were compared using one-way ANOVA with Tukey’s multiple comparisons test with asterisks indicating *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Results are from one experiment with n=3 per condition and are representative of two independent experiments. ADT, androgen deprivation therapy; ANOVA, analysis of variance; MDSC, myeloid-derived suppressor cell; TRT, targeted radionuclide therapy.

CXCR2 blockade improves antitumor efficacy in the TRT→ADT combination

Because tumor cells appeared primarily responsible for MDSC recruitment, and MDSC recruitment was inhibited in the presence of T cells, this suggested that CXCL1 and CXCL2 produced by tumor cells may be the dominant chemokines involved in MDSC recruitment. Consequently, we next tested if CXCL1 directly contributed to MDSC migration in vitro, and whether this might be affected by blockade of the CXCL1/CXCL2 receptor, CXCR2 (figure 7A). As demonstrated in figure 7B, we observed that MDSCs exhibited a migratory response toward supernatants containing CXCL1, and this response was significantly reduced when CXCR2 was blocked using reparixin. We next wanted to determine whether blocking CXCR2 could improve the antitumor response of the TRT→ADT treatment sequence (figure 7C). As demonstrated in figure 7D and online supplemental figure 11, mice treated with reparixin showed improved antitumor responses. Tumors from these mice exhibited a significant reduction in MDSCs (figure 7E), a slight increase in CD4+T cells (figure 7F), and a significant increase in CD8+T cells (figure 7G). Similar improved antitumor responses were found in mice treated with ADT→TRT and reparixin (online supplemental figure 12).