These authors contributed equally to this work.
This study aims to investigate the effect of splenectomy on radiation-mediated growth inhibition and immune modulation in lung cancer xenograft models.
Human non-small cell lung cancer H1299 cells and murine Lewis lung carcinoma LL/2-luc cells were injected into the right hind leg of BALB/c-nude mice and C57BL/6 mice, respectively. Splenectomy or sham operation was performed prior to tumor cell injection or before and after irradiation during tumor growth. Irradiation was delivered with 2–3 fractions of 6 Gy X-ray using a linear accelerator. Flow cytometry analysis was performed for immune cell profiling.
Splenectomy prior to tumor injection or at early stage inhibited growth of LL/2-luc tumors but not that of H1299 tumors; however, it did not enhance the antitumor effect of radiation regardless of intervention timing. Flow cytometry analysis showed monocytic myeloid-derived suppressor cells (MDSCs) and activated CD8+ T cells increased after irradiation in the tumors of splenectomized mice, compared to those of sham-operated mice. Administration of anti-PD-1 (programmed death-1) antibodies improved the ability of splenectomy to attenuate the growth of irradiated tumors.
Splenectomy has paradoxical effects on radiation-induced tumor growth inhibition, depending on tumor types and intervention timing, but it has an immune-modulating effect when combined with radiation.
Radiation therapy (RT), a major local treatment modality for cancer, exerts an array of immune modulatory effects [
The spleen, the largest secondary lymphoid organ, is a major site of extramedullary hematopoiesis and of MDSCs accumulation in cancers [
Murine lung carcinoma LL/2-Red-FLuc (LL/2-luc) cells were obtained from PerkinElmer (Waltham, MA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 g/mL streptomycin, 2 mM L-glutamine, and 25 mM HEPES. Cultures were maintained in a humidified atmosphere of 95% air/5% CO2 at 37°C. Human non-small cell lung cancer (NSCLC) H1299 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS, 100 U/mL penicillin, 100 g/mL streptomycin, 2 mM L-glutamine, and 25 mM HEPES.
All animal experiments were performed and approved in accordance with the Institutional Animal Care and Use Committee (IACUC) of Samsung Biomedical Research Institute (No. 20180625002, Approval date 2018-07-03; No. 20190926001, Approval date 2019-10-18). The animal study was carried out in compliance with the ARRIVE guidelines [
In BALB/c-nude mice, splenectomy or sham operation was performed prior to tumor cell injection. H1299 cells were harvested and suspended in a 1:1 ratio of phosphate-buffered saline (PBS) and Matrigel (Corning Inc., Tewksbury, MA, USA), and 1 × 106 cells/20 µL cells were injected subcutaneously into the right hind leg. Tumor volumes were calculated every 3 days with calipers according to the following formula:
When the mean tumor volume reached 500 mm3, tumors were irradiated with 2 fractions of 6 Gy X-ray on consecutive days (total dose of 12 Gy) to the right hind leg.
A LL/2-luc tumor model was generated by subcutaneously injecting LL/2-luc cells (1 × 106 cells/20 µL in 50% Matrigel) into the right hind leg in C57BL/6 mice. Splenectomy or sham operation was performed prior to tumor injection, at an early stage of tumor development (mean tumor volume of 200 mm3), or at an advanced stage of tumor growth (mean tumor volume of 1,000 mm3). Tumor irradiation began from the 7th day after tumor injection. Irradiation was given as 3 fractions of 6 Gy X-ray on consecutive days (total dose of 18 Gy). Mice in the treatment groups were intraperitoneally treated with anti-PD-1 (programmed death-1) antibody (2.5 mg/kg; Bio X Cell, Lebanon, NH, USA) on days 7, 9, 12, and 16.
Tumors implanted into mouse hind legs were irradiated as previously described [
Splenectomy or sham operation was performed under anesthesia induced by a sufficient amount of isoflurane using a liquid chamber for the vaporizer. Splenectomy was performed via a 0.5-cm skin incision made in the lateral abdomen and a midline fascia incision of the same length. The spleen was separated from the stomach and removed outside of the incision. The midline fascial incision was sutured with 4–0 thread and the skin incision was closed using 6–0 thread. The sham operation was performed by a skin incision and a midline fascia incision, in the same way as the splenectomy. However, the spleen remained inside the incision wound and the incision was closed. When surgeries were completed, mice were placed under a heat lamp and antibiotics (Baytril 50 inj., 5 mg/kg; Bayer, Leverkusen, Germany) and antiphlogistic drugs (Metacam 2%, 1 mg/kg; Boehringer Ingelheim, Ingelheim am Rhein, Germany) were injected intramuscularly for 3 days.
Tumors were harvested from the right hind leg, cut into small pieces, minced into a single-cell suspension, and mashed through a 70-μm cell strainer. Red blood cells were lysed with red blood cell lysing buffer (BD Bioscience, Franklin Lakes, NJ, USA).
Cell suspensions were stained with fluorescence-conjugated antibodies specific for CD45, CD11b, Ly6G, Ly6C, CD3, CD4, CD8, CD25 (BD Bioscience), and PD-L1 (eBioscience Inc., San Diego, CA, USA). For intracellular staining, cells were fixed with Fixation/Permeabilization buffer (eBioscience Inc.) and stained with anti-Foxp3 and anti-IFNγ (BD Bioscience) antibodies. Stained cells were analyzed by flow cytometry using a BD FACSVerse (BD Bioscience) and data analysis was performed using FlowJo software (BD Bioscience).
Tumors harvested were placed immediately into 10% neutral-buffered formalin (NBF). Tissues embedded in paraffin were sectioned at 4 μm. The tissue sections were stained with hematoxylin and eosin (H&E) for routine histological evaluation, and immunohistochemistry (IHC) was performed using the primary antibodies against CD3 (1:100 dilution), CD4 (1:1000 dilution), CD8 (1:2000 dilution; Abcam, Cambridge, UK) and Gr-1 (Ly6G/Ly6C, 1:100 dilution; Bio X Cell). Slides were digitally scanned with a digital pathology scanning system (Aperio ScanScope XT; Leica Biosystems, Buffalo Grove, IL, USA) and analyzed by using the ImageScope software (Leica Biosystems).
Statistical analyses were performed using GraphPad Prism 7 (Graphpad Software, San Diego, CA, USA). The significance of differences between experimental groups was calculated using one-way analysis of variance with Bonferroni’s test. The p-values <0.05 were considered statistically significant.
To understand a role of spleen, the largest lymphatic organ, in anti-tumor activity of radiation, LL/2 tumor-bearing legs were irradiated with a single fraction of 12 Gy or three fractions of 6 Gy (total 18 Gy), and spleens were collected either a day or 15 days after irradiation (
To evaluate how splenectomy affects radiation-induced tumor growth delay in a syngeneic LL/2 tumor model, splenectomy in C57BL/6 mice was performed prior to tumor injection (
Flow cytometric analyses revealed that there was no difference in the populations of neutrophils and M-MDSCs between splenectomized mice and sham-operated mice (upper panels in
To evaluate the effect of splenectomy on human lung tumor growth in immune-compromised mice, human NSCLC H1299 cells were implanted into hind legs of splenectomized BALB/c nude mice. H1299-tumor-bearing hind legs were irradiated with a total of 12 Gy of X-rays in two fractions (
Populations of neutrophils and PMN-MDSCs were not changed by radiation, splenectomy, or combination (
IHC analysis of Gr-1 (also Ly6C/Ly6G) showed that splenectomy significantly decreased Gr-1 positive cells in H1299 tumors with or without radiation (
Splenectomy was performed at two different time points after the injection of LL/2-luc tumors in C57BL/6 mice; at an early stage of tumor growth (mean tumor volume of 200 mm3), or at an advanced stage (mean tumor volume of 1,000 mm3) (
On the basis of the results that the combination of splenectomy and radiation increased tumor infiltration of PD-L1+ MDSCs (
In the present study, we aimed to investigate the effect of splenectomy on radiation-mediated tumor growth inhibition and immune modulation in a variety of settings. Radiation significantly decreased tumor burden in the syngeneic mouse model of lung cancer (
Our findings using splenectomized mice revealed that the anti-tumor effect of splenectomy is complex and context-dependent, as previously described by Prehn [
Immunophenotyping showed that intratumoral M-MDSCs increased after combination treatment with irradiation and splenectomy in both LL/2-luc and H1299 xenograft tumor models, although the anti-tumor effect of splenectomy in the two experiments was different (
A previous study using murine NSCLC cells by Levy et al. [
Synergistic antitumor effect of combined RT and anti-PD-L1 treatment has been well known, and reduced accumulation of MDSCs are also observed following this combination [
Our study has several limitations. The number of mice used was small, and flow cytometric analysis was performed at a single time point. Serial measurements of immune cell populations in more tumors at different time points might have given us more information regarding immunomodulatory effect of RT with or without splenectomy. Total dose of 18 Gy significantly attenuated tumor growth in all experimental conditions, which may make addition of splenectomy less effective. Thus, a further study is warranted to test the radiation dose-dependent effect on splenectomy.
Taken together, our data demonstrate that anti-tumor effect of splenectomy in tumor-bearing mice treated with radiation may be complex, depending on tumor types and intervention timing in lung cancer mouse models. These may be due to the extreme complexity of tumor-immune cell interactions within the microenvironment of different experimental settings. Our findings provide insight into the mechanism of immunomodulatory function of splenectomy during RT, such as an increase in activated T cells and PD-L1+ MDSC in tumors. Thus, our future studies will be directed to assess different non-invasive approaches to modulate the immunosuppressive effect of MDSCs and find optimal strategies of MDSC modulation to enhance radiotherapeutic or radio-immunotherapeutic effects.
No potential conflict of interest relevant to this article was reported.
This research was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (No. NRF-2017M2A2A7A02018569, NRF-2019R1F1A1061950).
Radiation inhibits growth of LL/2-luc tumors in a syngenic mouse model. (A) Experimental scheme. Murine Lewis lung carcinoma LL/2 cells were implanted into hind legs of C57BL/6 mice. The tumors were irradiated with either a single fraction of 12 Gy or 3 fractions of 6 Gy of X-rays. Splenectomy was performed one day or 15 days after irradiation (IR). (B) Tumor growth curves of LL/2 in mice. Radiation significantly reduced tumor growth, but there was little difference between two irradiation conditions. Data are presented as the mean ± standard error of the mean (n ≥ 4). All statistical analysis was performed using one-way analysis of variations (ANOVA) with a Bonferroni’s multiple comparisons test at the 5% level. ***p < 0.001, ****p < 0.0001.
Radiation modulates distribution of immune cells within the spleen of mice harboring LL/2-luc tumors. (A) Flow cytomtry analysis of neutrophils, M-MDSC, and PMN-MDSC in the spleens harvested one day or 15 days after irradiation (IR) to tumor sites. (B) Flow cytometry analysis of T lymphocytes in the spleens. Percentage of splenic CD4+ T cells, CD8+ T cells, regulatory T cells, and exhausted T cells was analyzed. Data are presented as the mean ± standard error of the mean (n ≥ 4). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. MDSC, myeloid-derived suppressor cells; M, monocytic; PMN, polymorphonuclear.
Splenectomy prior to tumor injection delayed tumor growth in a syngeneic LL/2-luc tumor model. (A) Experimental scheme. Splenectomy or sham operation was performed prior to tumor injection in C57BL/6 mice. The tumor-bearing mice were irradiated with 3 fractions of 6 Gy (tumor volume of 500 mm3). (B) Tumor growth curves of murine LL/2-luc cells in mice and (C) tumor volume at day 20 after tumor inoculation. In the splenectomy group, the tumor volume was decreased on day 20. Data are presented as the mean ± standard error of the mean (n ≥ 4). IR, irradiation. *p < 0.05, **p < 0.01.
Splenectomy enhanced the activation of tumor-infiltrating T lymphocytes in a LL/2-luc tumor model. Flow cytometry analysis of tumor-infiltrating (A) MDSCs and (B) T lymphocytes in a LL/2-luc tumor model. (A) M-MDSCs were increased in splenectomized mice with fractionated irradiation (IR). The population of PD-L1+ cells was also increased by irradiation. (B) IFNγ-releasing active CD8+ T cells were increased and Treg were decreased in splenectomized mice when combined with IR. Data are presented as the mean ± standard error of the mean (n = 4). MDSC, myeloid-derived suppressor cell; M, monocytic; PMN, polymorphonuclear; PD-L1, programmed death-ligand 1; INF, interferon. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Splenectomy prior to tumor injection had the limited effect on tumor growth in a human H1299 xenograft model. (A) Experimental scheme. Splenectomy or sham operation was performed prior to tumor injection in BALB/c-nude mice. H1299 tumor-bearing mice were irradiated with 2 fractions of 6 Gy (tumor volume of 500 mm3). (B) Tumor growth curves of H1299 in mice and (C) tumor volume at day 45 after tumor inoculation. Fractionated irradiation (IR) delayed tumor growth. Flow cytometry analysis of (D) neutrophils, (E) M-MDSC, and (F) PMN-MDSC frequency in tumors. M-MDSC are increased in splenectomized mice with fractionated IR. The percentage of PD-L1+ cells are also presented. Data are presented as the mean ± standard error of the mean (n ≥ 4). MDSC, myeloid-derived suppressor cell; M, monocytic; PMN, polymorphonuclear; PD-L1, programmed death-ligand 1. *p < 0.05, **p < 0.01, ****p < 0.0001.
Splenectomy modulated the infiltration of immune cells to tumor in H1299 and LL/2-luc tumor models. (A) Representative immunohistochemistry (IHC) images of Gr-1 in H1299 tumor tissues and (B) quantification. The mice bearing H1299 tumors were treated as described in
Splenectomy performed at an early stage of tumor growth had a tendency to inhibit tumor growth. (A) Experimental scheme. Splenectomy was performed at an early stage of tumor growth (tumor volume of 200 mm3) before irradiation (IR) or at an advanced stage (tumor volume of 1,000 mm3) after irradiation. (B) Tumor growth curves of LL/2-luc in mice and (C) tumor volume at day 20 after tumor inoculation. Splenectomy at an early stage (1st) but not that at the advanced stage (2nd) had a significant inhibitory effect on tumor growth. Data are presented as the mean ± standard error of the mean (n ≥ 4). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Administration of anti-PD-1 antibodies enhanced antitumor activity of IR in splenectomized mice. (A) Experimental scheme. Splenectomy was performed at an early stage of tumor growth. The LL/2-luc-tumor-bearing mice were irradiated with 3 fractions of 6 Gy and treated with an anti-PD-1 antibody on day 7, 9, 12, and 16. (B) Tumor growth curves of LL/2-luc in mice and (C) tumor volume at day 23 after tumor inoculation. Data are presented as the mean ± standard error of the mean (n ≥ 4). PD-1, programmed death-1; IR, irradiation. *p < 0.05, **p < 0.01, ****p < 0.0001.