Purpose:

The use of high-dose per fraction radiotherapy delivered as stereotactic body radiotherapy is a standard of care for prostate cancer. It is hypothesized that high-dose radiotherapy may enhance or suppress tumor-reactive immunity. The objective of this study was to assess both antitumor and immunosuppressive effects induced by high-dose radiotherapy in prostate cancer coclinical models, and ultimately, to test whether a combination of radiotherapy with targeted immunotherapy can enhance antitumor immunity.

Experimental Design:

We studied the effects of high-dose per fraction radiotherapy with and without anti-Gr-1 using syngeneic murine allograft prostate cancer models. The dynamic change of immune populations, including tumor-infiltrating lymphocytes (TIL), T regulatory cells (Treg), and myeloid-derived suppressive cells (MDSC), was evaluated using flow cytometry and IHC.

Results:

Coclinical prostate cancer models demonstrated that high-dose per fraction radiotherapy induced a rapid increase of tumor-infiltrating MDSCs and a subsequent rise of CD8 TILs and circulating CD8 T effector memory cells. These radiation-induced CD8 TILs were more functionally potent than those from nonirradiated controls. While systemic depletion of MDSCs by anti-Gr-1 effectively prevented MDSC tumor infiltration, it did not enhance radiotherapy-induced antitumor immunity due to a compensatory expansion of Treg-mediated immune suppression.

Conclusions:

In allograft prostate cancer models, high-dose radiotherapy induced an early rise of MDSCs, followed by a transient increase of functionally active CD8 TILs. However, systemic depletion of MDSC did not augment the antitumor efficacy of high-dose radiotherapy due to a compensatory Treg response, indicating blocking both MDSCs and Tregs might be necessary to enhance radiotherapy-induced antitumor immunity.

Translational Relevance

Stereotactic body radiotherapy (SBRT), a form of high-dose per fraction radiotherapy, has become a standard of care for prostate cancer because of its improved convenience and equivalent efficacy as compared with conventional radiotherapy. This is the first study to demonstrate that high-dose per fraction radiotherapy induces a strong, but transient antitumor immune response mediated by CD8 tumor-infiltrating lymphocytes, as well as an immunosuppressive response mediated by myeloid cells that could be therapeutically targeted to enhance the efficacy of SBRT in the treatment of prostate cancer.

Immune checkpoint inhibitors, which enhance antitumor immunity by removing the brakes on T-cell function, have led to a paradigm shift in the treatment of melanoma (1), lung cancer (2), renal cell cancer (3), and others. However, prostate cancer has proven to be poorly responsive to anti-program death-1 (anti-PD-1; ref. 3) and anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) therapies (4), although promising responses were reported in certain subsets of patients, including patients with microsatellite instability (5), biallelic loss-of-functional CDK12 (6), and possibly high tumor mutation burden (7–9). The mechanisms of resistance to immune checkpoint inhibition in prostate cancer are complex, including factors extrinsic to tumor cells that give rise to an immunosuppressive microenvironment, such as a paucity of tumor-infiltrating T lymphocytes, expression of a multitude of inhibitory immune checkpoints, and infiltration of various immunosuppressive cell populations (10). For example, in treatment-naïve prostate cancers, there is a lower density of CD8 cytotoxic T cells (11) and a higher fraction of CD4+ T regulatory cells (Treg; ref. 12). Tumor cells commonly express immune checkpoints, including program death-ligand 1 (PD-L1; ref. 13), and treatment with anti-PD-L1 antibody is shown to upregulate the expression of other inhibitory immune checkpoints, such as VISTA (14). In patients with prostate cancer, elevated levels of myeloid-derived suppressive cells (MDSC), particularly the monocytic-MDSC (M-MDSC) subset, in peripheral blood, are associated with a lower overall survival (15).

Radiotherapy, one of the first-line treatments for prostate cancer, is thought to exert its principal antitumor effect by inducing DNA damage (16). Beyond its cytotoxic effects, radiotherapy also induces a myriad of immunologic changes within the tumor microenvironment (17, 18). Briefly, radiation induces a proinflammatory response through multiple mechanisms, including promotion of antigen presentation, increased production of proinflammatory mediators, activation of dendritic cells, facilitation of antigen cross-presentation, modulation of immune checkpoint expression, and increased density of tumor-infiltrating lymphocytes (TIL; ref. 18). One of the well-delineated mechanisms involves the cGAS-STING pathway, which senses cytosolic DNA and activates type I IFN cascade to mediate adaptive immunity in response to radiation (19). Conversely, radiation also modulates elements of the immunosuppressive properties of tumor, such as increasing tumor infiltration or expansion of Tregs (20), tumor-associated macrophages (TAM; ref. 21), and MDSCs (21, 22). For example, local irradiation increases Treg infiltration (20), possibly through a substantial increase of the immunosuppressive cytokine TGFβ secretion (23). Irradiation of prostate tumors increases the production of multiple chemokines, such as colony-stimulating factor 1 and CXCL5, both of which drive recruitment of MDSCs and TAMs to tumors (21). Recent clinical data demonstrate that stereotactic body radiotherapy (SBRT) of localized high-risk prostate cancer induces a predominant shift toward myeloid cell infiltration (24). In addition, radiation-induced cGAS-STING activation, while on the one hand activating an antitumor immune response, can also trigger a downstream influx of M-MDSCs that drives radioresistance in tumors (25).

MDSCs are a heterogeneous population of immature cells of myeloid origin known for their immunosuppressive properties (26, 27). Signals generated from cancers or other pathologic conditions cause MDSC expansion and mobilize them to infiltrate blood, peripheral organs, and tumors (26). Abnormal expansion of MDSCs is related to overproduction of a variety of growth factors by tumors, such as GM-CSF, G-CSF, macrophage colony-stimulating factor (M-CSF), IL6, and VEGF and infiltration of MDSCs is attributable to an array of chemokines and their corresponding receptors, such as CXCL1/2/5/CXCR2, CXCL12/CXCR4, CCL2, S100A8, and S100A9 (26, 27). MDSCs exert their remarkable immunosuppressive ability via multiple mechanisms, including, but not limited to, depletion of nutrients l-arginine (28) and l-cysteine (29), necessary for lymphocyte activation, production of reactive oxygen and nitrogen species that affect antigen recognition and T-cell activation, induction of Tregs, reprogramming of macrophages, and promotion of angiogenesis (30). Interference with MDSC survival (31), recruitment (32, 33), and function (34, 35) has been demonstrated to improve the efficacy of radiation in murine models. Several clinical trials are currently investigating the impact of MDSC inhibitors on solid malignancies, such as RGX-104, YAP inhibitor, etc. (31, 32, 36). In this study, we chose to use Gr-1–neutralizing antibody because it was reported to significantly reduce malignant prostate weight and remarkably alter tumor histopathology in a spontaneous prostate cancer model (32).

SBRT allows precise delivery of extremely hypofractionated radiotherapy to tumor targets. Its use for prostate cancer has grown rapidly because of improved clinical convenience and equivalent efficacy as compared with conventional radiotherapy (37). To test the hypothesis that high-dose per fraction radiotherapy induces both robust antitumor CD8 TIL response and immunosuppressive myeloid cell infiltration, we studied CD8 TILs and MDSCs in syngeneic prostate cancer models using a high-dose per fraction regimen similar to that used in the clinic. We also evaluated the impact of MDSC depletion by anti-Gr-1 on tumor-reactive immune responses induced by radiotherapy.

Cell cultures

The RM-1 cell line was a gift from Dr. Johannes Czernin's laboratory (2017) and was originally obtained from the ATCC. The Myc-Cap cell line was a gift from Dr. William J. Aronson's laboratory (2017) and originally obtained from the ATCC. RM-1 and Myc-Cap cells were cultured in DMEM containing 10% FBS and 100 U/mL penicillin–streptomycin–glutamine at 37°C with 5% CO2. Cell lines were periodically authenticated by morphologic inspection and tested for Mycoplasma contamination by PCR tests, which were last performed in 2019. In general, cells with passage number 2–6 after thawing were used for experiments.

Mouse models and cell lines

C57BL6 and FVB/NCrlBR male mice (4–8 weeks old) were purchased from Charles River Laboratories. RM-1 (2.5 × 105) or Myc-Cap (2 × 106) cells were implanted subcutaneously in the thigh, and treatment was initiated when tumors became palpable, around 4–5 mm in diameter. All animal experiments were approved by the University of California, Los Angeles Institutional Animal Care and Use Committee (Los Angeles, CA) and conformed to all local and national animal care guidelines and regulations. Tumor size was measured by digital calipers every 2 days. Mice were sacrificed and tissues were analyzed at or before the ethical tumor size limit of 1.5 cm in diameter.

Local irradiation

Irradiation was performed using a Gulmay X-ray Machine (300 kV, 10 mA) with a dose rate of 1.705 Gy/minute. When tumors reached 4–5 mm in diameter, mice were anesthetized and irradiated with one (Myc-Cap) or two (RM-1) doses of 8 Gy to the tumor area with the rest of the body shielded. Two doses of 8 Gy were given 6 hours apart.

Flow cytometry

To prepare single-cell suspensions for flow cytometry, harvested tumors were dissected into approximately 1- to 3-mm (3) fragments and digested with 1 mg/mL Collagenase D (Sigma) and 0.1 mg/mL DNase I type IV (Sigma) in DMEM containing 10% FBS for 1 hour at 37°C while shaking gently. Spleens were gently dissociated using the back of a syringe for single-cell isolation. Peripheral blood was isolated directly into EDTA-coated Eppendorf tubes. Cells isolated from tissues were incubated with Fc block (anti-mouse CD16/32 antibody, eBioscience #14-0161-86) to prevent nonspecific binding. After red blood cell lysis (Lonza), single-cell suspensions were filtered through 40-μm filters and incubated for 30 minutes on ice with the following: FITC-Gr-1 (eBioscience, #11-5931-85), FITC-CD8 (eBioscience, #11-0081-86), FITC-CD44 (eBioscience, #11-0441-85), PE-Ly6C (eBioscience, #12-5932-82), PE-PD-1 (BioLegend, #135206), PE-CCR7 (eBioscience, #12-1971-82), PerCP-Cy5.5-CD3 (eBioscience, #45-0031-82), PerCP-Cy5.5-CD45 (BD Pharmingen, #550994), APC-CD11b (eBioscience, #17-0112-82), APC-CD8 (eBioscience, #17-0081-82), APC-CD62L (eBioscience, #17-0621-82), Pacific Blue-CD4 (eBioscience, #48-0042-82), eF450-Ly6G (eBioscience, 48-9668-82), eVolve655-CD8 (eBioscience, #86-0081-41), and eVolve655-CD45 (eBioscience, #86-0451-42). Cells were washed twice before analysis. Flow cytometry was performed using standard protocol on LSRII Analyzer (Becton Dickinson) and analyzed with FlowJo Software (Tree Star).

IHC

Tissues were harvested and fixed in 4% paraformaldehyde overnight. Sections (4 μm) were stained with the following antibodies: anti-Gr-1 (1:100; eBioscience), CD4 (Abcam, ab183685), CD3 (Agilent Technologies, A0452), and CD8 (eBioscience, 14-0808) antibodies. The samples were analyzed using an Aperio ScanScope AT digital scanner. Images were captured at 20× magnification, viewed using Aperio ImageScope v12.3.3, and analyzed using Definiens Tissue Studio 64 v4.3.0.

T-cell functional assay

Prostate tumor single cells were isolated by using the Mouse Tumor Dissociation Kit (Miltenyi Biotec). Debris were removed using Debris Removal Solution (Miltenyi Biotec). CD8 T cells were isolated using CD8 TIL Microbeads (Miltenyi Biotec) and loaded with Cell Tracer Violet (eBioscience). Isolated CD8 T cells were cultured with anti-CD3/CD28 (Thermo Fisher Scientific) and 30 U of recombinant mouse IL2 (eBioscience) for 3 days, or stimulated with PMA/Ionomycin (eBioscience) in the presence of GolgiPlug (BD Pharmingen) for 4.5–5 hours. Cells were stained with Zombie Nir (BioLegend), followed by surface marker staining with APC-CD8 (eBioscience). Cells were subsequently fixed and permeabilized using Fixation/Permeabilization Solution Kit (BD Pharmingen) and stained with FITC-IFNγ and PE-granzyme B following the manufacture's protocols.

MDSC depletion in vivo with anti-Gr-1 antibody

Anti-Gr-1 (clone RB6-8C5) and isotype control (clone LTF-2) were purchased from BioXCell and dosed at 200 μg/mouse (i.p.) every other day for the duration of the experiment. Tumor size were measure every other day using a caliper, and volume was calculated on the basis of the formula V = longest diameter × shortest diameter (2).

Statistical analysis

Data are presented as mean ± standard error of the difference (SED), unless indicated otherwise. Two-sided Student t test assuming two-tailed distributions was used to calculate statistical significance between groups. Prism (GraphPad) was utilized. P < 0.05 was considered statistically significant. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001.

High-dose per fraction radiotherapy induced a striking increase in CD4 and CD8 TILs

Tumors were generated by injecting 2.5 × 105 RM-1 cells, a Ras- and Myc-transformed murine prostate cancer cell line, into syngeneic C57/BL6 strain male mice (38). We irradiated the tumors when they became palpable and with a diameter of 4–5 mm (∼day 8 after inoculation). RM-1 tumor growth was significantly inhibited by two fractions of 8 Gy (Fig. 1A). This fractionation was selected because a single fraction of 8 Gy had minimal impact on tumor growth, whereas three fractions of 8 Gy completely suppressed tumor growth, leaving no tissue for assessment. Tumors and spleens were harvested at days 0, 2, 8, and 14 after radiation.

Figure 1.

Radiation induced a temporary increase of CD8 TILs in animals bearing RM-1 syngeneic prostate cancer. A total of 2.5 × 105 RM-1 cells were injected subcutaneously into left thigh, and tumors were irradiated with two fractions of 8 Gy when palpable, at around day 10. Tumors and spleens were harvested at days 0, 2, 8, and 14 after radiation. A, RM-1 tumor growth, size in mm3. B, Representative flow plots of CD4 and CD8 TILs at days (D) 2, 8, and 14 after radiation (gated on CD45+ cells). C and D, Bar plots showing the fractions of CD8 TILs and CD4 TILs in RM-1 tumors at days 2, 8, and 14 after radiotherapy (RT). E and F, Bar plots showing the fractions of CD8 TILs and absolute counts of CD8 TILs per gram of RM-1 tumor at day 8 after radiotherapy. G and H, Representative IHC images and bar plots of CD8 TILs in RM-1 tumors at day 8 after radiotherapy. I, Gating and representative plots of total memory, effector memory (EM), and central memory (CM) T cells in spleen. J, Bar plot showing fractions of splenic CD8 effector memory cell from RM-1 tumor–bearing animals at days 2, 8, and 14 after radiotherapy (n = 6–8/group). P < 0.05 was considered statistically significant. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Experiments were done four times. Ctrl, control.

Figure 1.

Radiation induced a temporary increase of CD8 TILs in animals bearing RM-1 syngeneic prostate cancer. A total of 2.5 × 105 RM-1 cells were injected subcutaneously into left thigh, and tumors were irradiated with two fractions of 8 Gy when palpable, at around day 10. Tumors and spleens were harvested at days 0, 2, 8, and 14 after radiation. A, RM-1 tumor growth, size in mm3. B, Representative flow plots of CD4 and CD8 TILs at days (D) 2, 8, and 14 after radiation (gated on CD45+ cells). C and D, Bar plots showing the fractions of CD8 TILs and CD4 TILs in RM-1 tumors at days 2, 8, and 14 after radiotherapy (RT). E and F, Bar plots showing the fractions of CD8 TILs and absolute counts of CD8 TILs per gram of RM-1 tumor at day 8 after radiotherapy. G and H, Representative IHC images and bar plots of CD8 TILs in RM-1 tumors at day 8 after radiotherapy. I, Gating and representative plots of total memory, effector memory (EM), and central memory (CM) T cells in spleen. J, Bar plot showing fractions of splenic CD8 effector memory cell from RM-1 tumor–bearing animals at days 2, 8, and 14 after radiotherapy (n = 6–8/group). P < 0.05 was considered statistically significant. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Experiments were done four times. Ctrl, control.

Close modal

To study changes in antitumor immune responses triggered by radiotherapy, we examined the density of tumor-infiltrating CD4 and CD8 T cells in the irradiated and control groups. Figure 1 shows the representative densities of CD4 and CD8 TILs at three separate timepoints after irradiation. Both CD4 and CD8 TILs were reduced by irradiation within 2 days (Fig. 1BD), likely representing apoptosis of preexisting TILs. However, 8 days after irradiation, there was a substantial increase of CD8 TILs in irradiated tumors (17 ± 5.8) compared with nonirradiated controls (2.8 ± 0.4), as detected by flow cytometry (Fig. 1B and C). The density of CD4 TILs in irradiated tumors was also doubled at day 8 after irradiation compared with nonirradiated controls (Fig. 1B and D). To ensure that the increase of CD8 TIL density was not merely due to a decrease in other immune cells, we calculated the absolute number of CD8 TILs in a separate experiment. The percentage of CD8 TILs among CD45+ leukocytes and the absolute CD8 TIL count per gram of tumor tissue was consistently elevated in irradiated tumors compared with controls (Fig. 1E and F). Similar results were observed when sections of tumors were examined by IHC (Fig. 1G and H), with significantly more CD8 T cells detectable in irradiated tumor tissue sections compared with nonirradiated controls. However, the increase in TILs was relatively short-lived and the density of both TIL populations dropped to baseline by day 14 (Fig. 1BD).

The changes in CD4 and CD8 TILs were also accompanied by an increase of memory T cells in the circulation (Fig. 1I and J). Figure 1I shows the gating and representative flow plots of total memory cells, effector memory, and central memory cells in the spleen. Eight days after radiation, there was a remarkable increase of CD8 effector memory cells, which remained elevated for at least another week (Fig. 1J). In summary, these results suggest that radiotherapy induced a strong, but transient local antitumor immune response, as well as immune memory in the systemic circulation in mice bearing syngeneic RM-1 prostate cancers.

Radiation-induced CD8 TILs were functionally competent

To investigate whether these radiation-induced CD8 TILs were functionally competent, we utilized two separate methods to evaluate CD8 TIL function. Our first method involved stimulating isolated CD8 TILs from tumors 8 days after radiotherapy in the presence of anti-CD3/CD28 beads and recombinant murine IL2 for 3 days. With this method, CD8 TILs isolated from irradiated tumors were significantly more proliferative, as indicated by the higher dilution of cell tracer (Fig. 2A and B). CD8 TILs from irradiated tumors also produced significantly more granzyme B, an effector molecule produced during CD8 T-cell degranulation (Fig. 2C and D), and IFNγ, a proinflammatory cytokine with important antitumor activity (Fig. 2E and F). Notably, Fig. 2E and F highlight a strikingly conspicuous population of CD8 TIL producing both granzyme B and IFNγ, which was only present in the irradiated tumors. These findings together demonstrate that radiotherapy not only induced an expansion of CD8 TILs, but also increased the number of CD8 TILs that were functionally active.

Figure 2.

Radiation (RT) induced a temporary increase of functional CD8 TILs in RM-1 tumors. Single-cell tumor suspensions from day 8 after radiation were used to isolate CD8 TILs, which were either cultured with anti-CD3/CD28 and recombinant mouse IL2 for 3 days, or stimulated with PMA/ionomycin for 5 hours. A and B, Representative histograms and bar plots of CD8 TIL proliferation. Cells were loaded with cell tracer violet before stimulation with anti-CD3/CD28 beads, and the proliferation was calculated on the basis of the titration of the cell tracer. Cells that underwent more division had lower tracer intensity. C and D, Representative flow plots and bar plot of granzyme B (GrzB) production by CD8 TILs. E and F, Representative flow plots and bar plot of IFNγ and granzyme B production by CD8 TILs. G, Bar plot showing radiation did not alter the percentage of CD8 TILs producing IFNγ. H and I, Bar plots showing increase in the density and absolute numbers of IFNγ-producing CD8 TILs. J, Bar plot showing the percentage of PD-1 expression on CD8 TILs at days 2 and 8 after irradiation (n = 6–8 mice/group). P < 0.05 was considered statistically significant. *, P < 0.05; **, P < 0.005. Experiments were done twice. Ctrl, control.

Figure 2.

Radiation (RT) induced a temporary increase of functional CD8 TILs in RM-1 tumors. Single-cell tumor suspensions from day 8 after radiation were used to isolate CD8 TILs, which were either cultured with anti-CD3/CD28 and recombinant mouse IL2 for 3 days, or stimulated with PMA/ionomycin for 5 hours. A and B, Representative histograms and bar plots of CD8 TIL proliferation. Cells were loaded with cell tracer violet before stimulation with anti-CD3/CD28 beads, and the proliferation was calculated on the basis of the titration of the cell tracer. Cells that underwent more division had lower tracer intensity. C and D, Representative flow plots and bar plot of granzyme B (GrzB) production by CD8 TILs. E and F, Representative flow plots and bar plot of IFNγ and granzyme B production by CD8 TILs. G, Bar plot showing radiation did not alter the percentage of CD8 TILs producing IFNγ. H and I, Bar plots showing increase in the density and absolute numbers of IFNγ-producing CD8 TILs. J, Bar plot showing the percentage of PD-1 expression on CD8 TILs at days 2 and 8 after irradiation (n = 6–8 mice/group). P < 0.05 was considered statistically significant. *, P < 0.05; **, P < 0.005. Experiments were done twice. Ctrl, control.

Close modal

We also evaluated CD8 TIL function by combining single-cell suspensions with PMA/ionomycin for 5 hours, followed by evaluation of IFNγ production. Irradiation did not change the ability of individual CD8 TILs to produce IFNγ (Fig. 2G). However, because there were significantly higher numbers of CD8 TILs within the irradiated tumors, the percentage and absolute number of IFNγ-producing CD8 TILs were remarkably increased after irradiation (Fig. 2H and I). Interestingly, CD8 TILs from the irradiated tumors also had increased PD-1 expression over time (Fig. 2J). This suggests a model where irradiation increases the total number of functional CD8 TILs that start to become functionally exhausted with increased PD-1 expression, which correlates with their subsequent depletion within the irradiated tumors.

High-dose per fraction radiotherapy induced an immediate increase and a subsequent drop of tumor-infiltrating MDSCs

To investigate what immunosuppressive changes might be induced by high-dose per fraction radiotherapy, we focused on MDSCs. MDSCs are reported to increase gradually after conventionally fractionated radiotherapy (21). Figure 3A shows the gating strategy for tumor-infiltrating MDSC (CD11b+Gr-1+) and its subsets, M-MDSC (Ly6C+Ly6G) and polymorphonuclear-MDSC (PMN-MDSC; Ly6CLy6G+). Figure 3B and C demonstrate that high-dose per fraction radiotherapy induced an immediate increase of tumor-infiltrating MDSCs at day 2 after irradiation, which was much earlier than the published data for conventionally fractionated radiation models, typically 5 days after the completion of radiation. Following the sudden increase of MDSCs, there was a transient decrease of MDSCs at day 8 after irradiation, which coincided with the expansion of CD8 TILs. This drop in MDSCs has not been reported in published conventionally fractionated models. However, this reduction of MDSCs was short-lived, as their level increased again to above baseline at day 14 after irradiation. Further characterization of the MDSC subsets revealed that the day 2 increase and day 8 reduction of MDSCs were both largely driven by the M-MDSC subset (Fig. 3D), while the day 14 was largely comprised of PMN-MDSCs (Fig. 3E).

Figure 3.

High-does per fraction radiotherapy (RT) temporarily increased tumor-infiltrating MDSCs in RM-1 and Myc-Cap syngeneic tumors. RM-1: RM-1 tumors were irradiated with two fractions of 8 Gy when palpable, at around day 10, and harvested at days 2, 8, and 14 after irradiation. A, Flow plots showing the gating strategy for MDSCs and its subsets. Total MDSCs (Gr-1+CD11b+), M-MDSCs (Ly6C+Ly6G), and PMN-MDSCs (Ly6CLy6G+). B, Representative flow plots of total MDSCs within irradiated and nonirradiated RM-1 tumors at days 2, 8, and 14 after radiation. CE, Bar plots of intratumoral total MDSCs, M-MDSCs, and PMN-MDSCs at days 2, 8, and 14 after irradiation. Because of the rapid growth of nonirradiated RM-1 tumors, the animals were sacrificed before day 14 after radiotherapy (n = 6–8 mice/group). Myc-Cap: A total of 2 × 106 Myc-Cap cells were injected subcutaneously into left thigh. Tumors were irradiated with a single fraction of 8 Gy when palpable, at around day 12. F, Myc-Cap tumor growth (mm3) with or without irradiation. G, Representative flow plots of total MDSCs from Myc-Cap tumors at days 2, 10, and 20 after the date of radiotherapy (n = 6–8 mice/group). P < 0.05 was considered statistically significant. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Experiments were done four times. Ctrl, control.

Figure 3.

High-does per fraction radiotherapy (RT) temporarily increased tumor-infiltrating MDSCs in RM-1 and Myc-Cap syngeneic tumors. RM-1: RM-1 tumors were irradiated with two fractions of 8 Gy when palpable, at around day 10, and harvested at days 2, 8, and 14 after irradiation. A, Flow plots showing the gating strategy for MDSCs and its subsets. Total MDSCs (Gr-1+CD11b+), M-MDSCs (Ly6C+Ly6G), and PMN-MDSCs (Ly6CLy6G+). B, Representative flow plots of total MDSCs within irradiated and nonirradiated RM-1 tumors at days 2, 8, and 14 after radiation. CE, Bar plots of intratumoral total MDSCs, M-MDSCs, and PMN-MDSCs at days 2, 8, and 14 after irradiation. Because of the rapid growth of nonirradiated RM-1 tumors, the animals were sacrificed before day 14 after radiotherapy (n = 6–8 mice/group). Myc-Cap: A total of 2 × 106 Myc-Cap cells were injected subcutaneously into left thigh. Tumors were irradiated with a single fraction of 8 Gy when palpable, at around day 12. F, Myc-Cap tumor growth (mm3) with or without irradiation. G, Representative flow plots of total MDSCs from Myc-Cap tumors at days 2, 10, and 20 after the date of radiotherapy (n = 6–8 mice/group). P < 0.05 was considered statistically significant. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Experiments were done four times. Ctrl, control.

Close modal

Next, we investigated whether high-dose per fraction radiotherapy similarly induced an immunosuppressive environment in other syngeneic prostate cancer models. We chose the cMyc-overexpressing Myc-Cap cell line that is androgen dependent and is syngeneic to the FVB strain. We injected 2 × 106 Myc-Cap cells s.c. into the left thigh, and irradiated the tumors when they became palpable and 4–5 mm in diameter (∼12 days) after tumor inoculation. We observed that two fractions of 8 Gy resulted in near tumor eradication (Supplementary Fig. S1); therefore, we used a single fraction of 8 Gy for the Myc-Cap tumor model, which resulted in tumor suppression, but not eradication (Fig. 3F). Following a similar protocol, we harvested tumors from both irradiated and nonirradiated groups at days 2, 10, and 20 after irradiation. A single fraction of 8 Gy rapidly decreased CD8 TILs 2 days after radiation, but at 10 and 20 days after radiation, CD8 TILs were not significantly different between radiotherapy and control group (Supplementary Fig. S2), indicating the antitumor immune response was not as predominant as in RM-1 model. In nonirradiated controls, tumor-infiltrating MDSCs naturally increased steadily overtime (Fig. 3G), yet radiation induced a significant increase of total MDSCs 2 days after irradiation, followed by a significant reduction at day 10 (Fig. 3G; Supplementary Fig. S3A). At day 20 after irradiation, both groups had a remarkable increase of MDSCs, the majority of which were PMN-MDSCs (Supplementary Fig. S3B and S3C). This finding is consistent with the observation in other tumors that PMN-MDSC density is proportionate to tumor volume (31). In Myc-Cap tumors, the radiation-induced MDSC surge at day 2 was largely driven by PMN-MDSCs, with very few M-MDSCs in the tumor at that time. The data from these two syngeneic models demonstrate that radiation induced an immediate increase of MDSCs, followed by a transient reduction preceding a final increase in MDSCs. Given that MDSCs inhibit CD8 cell activation and expansion (31, 39), we hypothesized that inhibition or depletion of the MDSCs may enable a more durable and robust CD8 TIL increase in density and activity after irradiation.

Neutralization of Gr-1 depleted systemic MDSCs and inhibited MDSC tumor infiltration

To determine whether further reduction of MDSCs in the setting of high-dose per fraction radiotherapy would permit a more long-lasting effect on antitumor immunity, we set out to deplete MDSCs using a Gr-1–neutralizing antibody in our syngeneic prostate tumor models. Anti-Gr-1 has been reported to reduce MDSCs in peripheral blood after one dose, and completely eliminate MDSCs in peripheral blood and tumors in just a few weeks (32). Figure 4A shows our experimental design. We injected anti-Gr-1 (clone RB6-8C5) or isotype control (clone LTF-2) to RM-1 tumor–bearing animals at 200 μg per mouse every other day for 3 days, irradiated tumors at day 8, and harvested blood, spleen, and tumor at days 10 (2 days after radiotherapy), 16 (8 days after radiotherapy), and 20 (12 days after radiotherapy). We observed that two doses of anti-Gr-1 already effectively eradicated MDSCs in peripheral blood (Supplementary Fig. S4A) and reduced splenic MDSCs to about 1% of its control level (Supplementary Fig. S4B) on day 10 (2 days after radiation). Similarly, anti-Gr-1 significantly decreased MDSC infiltration within tumors (Fig. 4B, top; Supplementary Fig. S4C). Given Gr-1 is a marker for MDSCs, we wanted to rule out the possibility that MDSCs might not be detected because of the blocking effect of anti-Gr-1; therefore, we evaluated the percentage of M-MDSCs and PMN-MDSCs gated on CD45+CD11b+ myeloid cells, both of which were significantly reduced as well (Fig. 4B, bottom). These results demonstrate the ability of anti-Gr-1 to effectively deplete MDSCs in systemic circulation and tumors.

Figure 4.

Anti-Gr-1 (αGr-1) did not enhance antitumor immunity due to the induction of compensatory Treg response. Anti-Gr-1 (clone RB6-8C5) or isotype control (clone LTF-2) was injected into RM-1 tumor–bearing animals at 200 μg per mouse every other day. Blood, spleens, and tumors were harvested at tumor days 10 [2 days after radiotherapy (RT)], 16 (8 days after radiotherapy), and 20 (12 days after radiotherapy). A, Experimental design. B, Bar plots of intratumoral MDSCs and its subsets at day 10 after tumor inoculation (2 days after radiotherapy; n = 3–4 mice/group). C, RM-1 tumor growth until tumor day 18 (day 12 after radiotherapy). Open square, no radiotherapy + IgG; open circle, radiotherapy + IgG; solid square, no radiotherapy + anti-Gr-1; and solid circle, radiotherapy + anti-Gr-1. D, Percentage of CD8 TILs at tumor day 16 (8 days after radiotherapy). E, Percentage of IFNγ-producing CD8 TILs at tumor day 16 (8 days after radiotherapy). F, Percentage of granzyme B-producing CD8 TILs at tumor day 16 (8 days after radiotherapy). G, Percentage of CD4 TILs at tumor day 16 (8 days after radiotherapy). Hand I, Fractions of CD4 TILs and Tregs at tumor day 20 (day 12 after radiotherapy; n = 4–8 mice/group). P < 0.05 was considered statistically significant. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001. Experiments were done twice. i.p., intraperitoneal.

Figure 4.

Anti-Gr-1 (αGr-1) did not enhance antitumor immunity due to the induction of compensatory Treg response. Anti-Gr-1 (clone RB6-8C5) or isotype control (clone LTF-2) was injected into RM-1 tumor–bearing animals at 200 μg per mouse every other day. Blood, spleens, and tumors were harvested at tumor days 10 [2 days after radiotherapy (RT)], 16 (8 days after radiotherapy), and 20 (12 days after radiotherapy). A, Experimental design. B, Bar plots of intratumoral MDSCs and its subsets at day 10 after tumor inoculation (2 days after radiotherapy; n = 3–4 mice/group). C, RM-1 tumor growth until tumor day 18 (day 12 after radiotherapy). Open square, no radiotherapy + IgG; open circle, radiotherapy + IgG; solid square, no radiotherapy + anti-Gr-1; and solid circle, radiotherapy + anti-Gr-1. D, Percentage of CD8 TILs at tumor day 16 (8 days after radiotherapy). E, Percentage of IFNγ-producing CD8 TILs at tumor day 16 (8 days after radiotherapy). F, Percentage of granzyme B-producing CD8 TILs at tumor day 16 (8 days after radiotherapy). G, Percentage of CD4 TILs at tumor day 16 (8 days after radiotherapy). Hand I, Fractions of CD4 TILs and Tregs at tumor day 20 (day 12 after radiotherapy; n = 4–8 mice/group). P < 0.05 was considered statistically significant. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001. Experiments were done twice. i.p., intraperitoneal.

Close modal

Anti-Gr-1 treatment did not enhance antitumor immunity due to the induction of a Treg response

Next, we investigated whether depletion of MDSCs would enhance radiation-induced antitumor immunity. As expected, radiotherapy significantly inhibited tumor growth regardless of anti-Gr-1 treatment (Fig. 4C). Unexpectedly, in the absence of radiotherapy, anti-Gr-1 treatment resulted in more rapid tumor growth as compared with IgG control. In addition, anti-Gr-1 did not add to any additional tumor growth inhibition when combined with radiotherapy (Fig. 4C). This unexpected result suggest that although anti-Gr-1 successfully eradicated MDSCs, it did not enhance tumor control by radiotherapy.

We next examined the CD8 TIL populations across the four groups at day 16 (8 days after radiotherapy). The groups receiving radiotherapy with or without anti-Gr-1 showed no significant difference in the percentage of CD8 TILs, although both were significantly higher than their respective nonirradiated controls (Fig. 4D). Similarly, the density of IFNγ-producing CD8 TILs was comparable between the two groups receiving radiotherapy (Fig. 4E). These data confirmed that anti-Gr-1 did not enhance a radiotherapy-induced CD8-mediated immune response. Interestingly, the density of IFNγ-producing CD8 TILs was significantly lower in the group receiving anti-Gri-1 alone compared with IgG alone (Fig. 4E), indicating anti-Gr-1 might cause a reduction of functional CD8 TILs independent of irradiation.

We also observed that anti-Gr-1 treatment alone increased PD-1 expression on CD8 TILs, and that combined treatment of anti-Gr-1 and radiotherapy increased PD-1 expression on CD8 TILs compared with radiotherapy alone (Supplementary Fig. S5A). The increase of PD-1 expression of CD8 TILs was not directly correlated with the status of exhaustion, as there was a significant fraction of PD-1+ CD8 TILs that were concomitantly producing IFNγ (Supplementary Fig. S5B). Next, we wanted to find out whether cytotoxic activity of the CD8 TILs was altered by anti-Gr-1 treatment. We isolated CD8 TILs from tumors harvested at day 8 after irradiation, and analyzed their production of granzyme B after stimulation with anti-CD3/CD28 for 3 days. The amount of granzyme B produced by the CD8 TILs after anti-Gr-1 treatment was significantly lower compared with IgG control (Fig. 4F), indicating there was indeed functional impairment of CD8 TILs by anti-Gr-1 treatment.

To understand what factors might be associated with the reduction of CD8 TIL degranulation after anti-Gr-1 treatment, we quantified the density of CD4 TILs in the tumors. Surprisingly, anti-Gr-1 increased the density of CD4 TILs in the anti-Gr-1 groups both with and without radiotherapy compared with their respective IgG controls (Fig. 4G). Four days later, at day 12 after radiotherapy, we observed a continued elevation of total CD4 TILs, as well as Tregs in the anti-Gr-1 treatment group compared with IgG control (Fig. 4H and I), indicating that depletion of MDSCs by anti-Gr-1 led to an expansion of Tregs. These results together suggest that MDSC depletion by anti-Gr-1 treatment induced a compensatory expansion of Treg-mediated immunosuppression, which may explain the increased tumor growth in the animals receiving anti-Gr-1. In addition, this compensatory Treg expansion did not inhibit CD8 TIL infiltration or their ability to produce proinflammatory cytokine IFNγ, but did decrease the ability of CD8 TILs to degranulate, an important part of CD8 cytotoxicity and antitumor machinery.

Although a number of groups have reported on radiation-induced immune changes in various animal models (20, 21), our understanding of the role of high-dose per fraction radiotherapy, particularly in the setting of prostate cancer and relevant coclinical models, remains limited. In this study, we used two murine syngeneic prostate cancer models to study the local and systemic immune responses induced by high-dose per fraction radiotherapy, and established that the radiotherapy induced both time-dependent antitumor and immunosuppressive responses.

There is considerable evidence showing local radiation induces immunologic changes in the tumor microenvironment (18). Previous studies reported that five fractions of 3 Gy induced myeloid cell infiltration in a prostate tumor model (21). Likewise, a single dose of 20 Gy induced both MDSC and CD8 T-cell infiltration in a colon cancer model (22), and a single dose of 10 Gy increased Tregs within murine models of melanoma, renal, and colon cancers (20). Therefore, different radiation doses and fraction sizes may alter the extent of antitumor versus immunosuppressive responses. The fraction size, 8 Gy, chosen for this study is commonly used in the clinic for prostate SBRT. Moreover, this fraction size has been shown to induce a strong CD8 T-cell response while inducing minimal Treg increase in prior murine models of other cancer types (40).

In this study, we showed that high-does per fraction radiotherapy induced a rapid infiltration of MDSCs followed by a striking, but transient expansion of CD8 TILs corresponding to the nadir of the MDSCs, confirming the hypothesis that high-dose per fraction radiotherapy simultaneously induces antitumor immunity along with an immunosuppressive response, at different timepoints. The numerical increase of CD8 TILs after radiation is not only attributable to increased tumor infiltration, but also to their active proliferation (Fig. 2A). In addition, these radiotherapy-induced CD8 TILs were more functionally competent than their controls, as demonstrated by their ability to secret IFNγ and degranulate (Fig. 2CF). However, this promising antitumor immune response was transient. The CD8 TILs rapidly diminished within a few days.

M-MDSCs and PMN-MDSCs are phenotypically similar to monocytes and neutrophils, respectively (27). While both subsets use arginase-1 for their suppressive activities, PMN-MDSCs produce higher levels of reactive oxygen and nitrogen species (41), and M-MDSCs produce higher amounts of NO, consume more T-cell–dependent nutrients, and induce more Treg differentiation (42). In addition, compared with PMN-MDSCs, M-MDSCs are more proliferative (43) and retain the ability to differentiate into immunosuppressive macrophages (44) and PMN-MDSCs (43) within the tumor. These attributes of MDSCs are consistent with our observation in RM-1 tumor that M-MDSC is the predominant subset to rise early after radiation, and the subsequent increase in PMN-MDSCs could represent either delayed infiltration or differentiation from M-MDSCs. In both syngeneic coclinical models, we consistently observed an early rise of MDSCs at day 2 after radiotherapy, and a second increase 2 weeks after radiation, mainly comprised of PMN-MDSCs. We also observed that total MDSCs and M-MDSCs were consistently and significantly reduced at day 8 after radiation, coinciding with a concurrent expansion of functional CD8 TILs in the RM-1 model. The first wave of MDSCs was largely driven by M-MDSCs in the RM-1 model and by PMN-MDSCs in the Myc-Cap model. The different infiltration patterns of MDSC subsets may be the result of different chemokine and cytokine profiles induced by radiotherapy in these two tumors. Further investigation of radiotherapy-induced inflammatory mediators and the biology of MDSC subsets may shed light on the mechanism of differential infiltration.

To determine whether suppression of MDSC tumor infiltration would result in persistence of the increase in CD8 TILs, thereby increasing the durability of the antitumor immunity induced by high-dose per fraction radiotherapy, we blocked systemic and intratumoral MDSCs using Gr-1–neutralizing antibody. Unexpectedly, anti-Gr-1 failed to decrease tumor growth either alone or in combination with radiotherapy (Fig. 4C). Combined with radiation, anti-Gr-1 did not change the density of CD8 TILs or their ability to produce IFNγ, but did reduce their production of granzyme B. Hypothesizing that other immunosuppressive mechanisms might have been stimulated upon MDSC eradication, we examined the changes in CD4 TILs after anti-Gr-1 treatment. We discovered that anti-Gr-1 significantly increased the density of CD4 TILs irrespective of radiation (Fig. 4G and H). Not surprisingly, Tregs were significantly increased after anti-Gr-1 treatment, indicating that depletion of MDSCs with anti-Gr-1 induced a compensatory expansion of Treg that resulted in a net protumor activity. Indeed, PMN-MDSCs have been shown to suppress TGFβ1-induced generation of FoxP3+ Tregs via inhibition of early T-cell activation, and this process is dependent on reactive oxygen species and indoleamine 2,3-dioxygenase (45). Therefore, we hypothesized that depletion of PMN-MDSCs removes the inhibition of Treg differentiation and results in dis-inhibition of Treg-mediated immunosuppressive responses.

The unique aspect of this study is that in syngeneic allograft prostate cancer models, high-dose per fraction radiotherapy induced a complex interaction of different immune populations. It is promising that high-dose per fraction radiotherapy increases highly functional CD8 TILs 8 days after irradiation, which coincides with a drop of total MDSCs, mainly driven by the M-MDSC subset. However, this transient wave of CD8 TILs is negated by the expansion of PMN-MDSCs 7–10 days later, which appears to be intrinsic to prostate tumors and independent of radiation. Gr-1 neutralization successfully eradicated MDSCs, but induced a compensatory increase of tumor-infiltrating Tregs, resulting in a net inhibitory effect on CD8 TILs and a negligible effect on radiation-induced tumor growth suppression. These results together suggest that effective use of radiotherapy-induced immunity to eradicate tumors will require combined approaches to suppress radiotherapy-induced protumor immunity to tip the balance more completely to an antitumor response. Possible opportunities include simultaneous blockade of MDSC and Treg populations, or the use of checkpoint inhibitors to augment CD8 TIL activity with or without cotargeting of suppressor cells (46). We have recently completed a clinical trial of SBRT neoadjuvant to radical prostatectomy in patients with high-risk prostate cancer using fraction sizes of 8 Gy (47). We are evaluating the pre- and posttreatment tissue to discover what immunologic changes take place post-SBRT in patients to confirm the diverse immune responses seen in our preclinical models. We plan to synthesize this coclinical and clinical data to design optimized combined modalities leveraging SBRT with immunotherapy to target both local and metastatic tumor deposits.

In summary, we demonstrated that high-dose per fraction radiotherapy induced a complex, time-dependent change in the tumor microenvironment in syngeneic prostate cancer models, characterized by both dynamic changes in infiltrating MDSC subsets and reactive CD8 TILs. Radiotherapy induced a transient surge in reactive CD8 TILs that was not enhanced by anti-Gr-1–mediated depletion of MDSCs, likely due to compensatory expansion of suppressive Tregs. A logical strategy would include cotargeting Tregs. In addition, checkpoint inhibition, including targeting PD-L2 (48), which was expressed at higher level than PD-L1 in prostate cancer and strongly associated with radiation response, represents a complementary strategy to counteract immunosuppression within the postradiation tumor microenvironment. Future studies are warranted to investigate the effects of cotargeting of MDSCs with Tregs or checkpoint inhibition in the setting of high-dose per fraction radiotherapy on prostate tumors.

N.G. Nickols reports grants from Prostate Cancer Foundation, UCLA Prostate SPORE, and UCLA Jonsson Comprehensive Cancer Center during the conduct of the study, and grants from Progenics Pharmaceuticals, Janssen, Baye, and VA ORD outside the submitted work. No disclosures were reported by the other authors.

L. Lin: Conceptualization, data curation, software, formal analysis, visualization, methodology, writing-original draft, writing-review and editing. N. Kane: Software, methodology. N. Kobayashi: Software, validation, methodology. E.A. Kono: Methodology. J.M. Yamashiro: Resources, methodology. N.G. Nickols: Conceptualization, resources, supervision, project administration, writing-review and editing. R.E. Reiter: Conceptualization, resources, supervision, project administration, writing-review and editing.

This work was supported by Young Investigator Award from the Prostate Cancer Foundation (to N.G. Nickols), a Research Career Development Award from STOP CANCER; SPORE P50 in Prostate Cancer (to R.E. Reiter); and S Anne P. & Robert A. Williams, Jr. Fund with the Arizona Community Foundation (to R.E. Reiter). We thank Drs. Johannes Czernin and William J. Aronson for gifting us the RM-1 and Myc-Cap cell lines, respectively. We thank UCLA Flow Core, Division of Laboratory Animal Medicine, Radiation Oncology X-ray facility, and Radiation Oncology Vivarium for their service.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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