Animal tissue research was carried out in accordance with the requirements of the Shandong University Human and Animal Ethics Research Committee approval 20140315. The breast cancer model in rats has been described previously. In brief, after the model was successfully established on or about the 90th day after a single intragastric administration of DMBA, the tumors accounted for approximately 10% of the total animal weight. Two or more tumors were randomly assigned to four different groups: the control group, HPTA group alone, RT group alone, and RT + HPTA group. HPTA was administered intraperitoneally (20 mg/kg). RT (PRECISION X-Rad 225, USA; dose fractionation 2 Gy/time/day) of the tumor was in situ. The HPTA concentration was selected after a series of pre-clinical toxicology experiments were conducted, and the radiation dose and dose fractionation strategy used in this study was also based on previous research[21, 29-32]. The specific details of HPTA and RT treatments are shown in Table 1. The tumor size was monitored weekly and measured with vernier calipers to determine the tumor response until day 35. Tumor volume was calculated according to the clinical standard formula as follows:
Time 1st Day 2nd Day 3rd Day 4th Day 5th Day 6th Day 8:00 am HPTA HPTA HPTA HPTA HPTA HPTA 14:00 pm − 2 Gy 2 Gy 2 Gy 2 Gy − 20:00 pm HPTA HPTA HPTA HPTA HPTA HPTA Note. RT: Radiotherapy. HPTA: 2-hexyl-4-pentylenic acid.
Table 1. Schedule of HPTA and RT treatment for rats in the RT + HPTA treatment group
tumor volume (mm3) = Length (L) × Width (W)2/2.
The tissue was collected on day 15, and the mammary tumors were isolated and mechanically separated into small tissue pieces, as previously described[26, 28]. The morphological structure of the tumor tissue was observed by performing HE staining (Figure 1B and Figure 2A).
Figure 1. RT induces an abscopal effect in vivo when combined with HPTA. (A) Representative pictures of the normal breast tissue (1) and DMBA-induced breast cancer tissue established in rats (2 and 3). (B) The morphological structure of breast normal tissue (4) and DMBA-induced breast cancer tissue (5) by HE staining. The pathological changes are indicated by black arrows. (C) The schedule of the studies, including DMBA treatment, HPTA administration, RT treatment, and analysis. (D) The changes of the standardized tumor volume within 35 days after RT and HPTA treatment. Each data point in the graph was from three independent experiments (mean ± SD).
Figure 2. The combination of HPTA and RT can inhibit the proliferation of tumor cells and promote the infiltration of myeloid-derived macrophages. (A) The morphology of tumor tissues in the control, HPTA-only, RT-only, and RT + HPTA groups by HE staining. The pathological changes are marked with black arrows. (B) IHC analysis was performed using the markers BrdU and Ki67 to evaluate tumor proliferation and are presented in the photographs (left). The relative amount of BrdU and Ki67 positive cells was further quantified using Image-Pro Plus software by IOD, as shown in the graphs (right). The areas of immunostaining are indicated by black arrows. (C) IHC staining was performed using the specific cell markers F4/80 and CD68 and presented in the photographs (left). The macrophages are indicated by black arrows. The relative amount of F4/80 and CD68 positive cells was further quantified (right). (D) IHC staining was performed using the myeloid cell marker CD11b and the relative amount of CD11b positive cells was presented in the graph (right). (E) Co-staining of immunofluorescence with the markers CD11b and F4/80 was performed to identify infiltrated myeloid-derived F4/80+ macrophages (left). Co-localization was observed in the merged pictures (marked with white arrows). The ratio of myeloid-derived F4/80+ in total F4/80+ macrophages is shown in the graph (right). The data in the graph were obtained from three independent experiments (mean ± SD, *P < 0.05, **P < 0.01).
The resected tumors were fixed in 10% formalin overnight, embedded in paraffin, and sectioned. The sections were dewaxed with xylene and hydrated using a graded series of ethanol solutions from 100% to 75% ethanol. Antigen exposure was conducted by heating the sections in 10 mmol/L sodium citrate buffer for 20 min at a water bath temperature of 92 °C. The sections were cooled and incubated in 3% hydrogen peroxide for 15 min at room temperature. Antigen blocking was carried out by incubating with 10% goat serum for 1 h and then staining in block buffer containing primary antibody diluted in TBS with 1% BSA overnight at 4 °C in the dark. The primary antibodies used were: anti-BrdU (BD, 0029341, 1:50), anti-Ki67 (Cell Signaling, 12202S, 1:400), anti-F4/80 [BioLegend, 123102, 1:100 (IHC), 1:300 (IF)], anti-CD68 (Servicebio, GB11067, 1:1000), anti-CD11b antibody [Abcam, ab133357, 1:4000(IHC), 1:1000(IF)], anti-CD86 [Invitrogen, 3100F4A7, 1:100(IHC, IF)], anti-CD31 [Servicebio, GB12063, 1:200(IF)], and anti-CD34 [Santa Cruz Biotechnology, Sc-74499, 1:100(IF)]. The secondary antibodies used were: goat anti-rabbit IgG (1:300, BA-1000, Vector), goat anti-mouse IgG (1:300, BA-9200, Vector), and goat anti-rat IgG (1:300, BA-9400, Vector). IHC quantitation was performed using Image-Pro Plus 4.5 software (Media Cybrenetics, Silver Spring, USA) and expressed by integral optical density (IOD).
Immunofluorescence method is detailed elsewhere. The secondary antibodies used were: Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, 34732A, 1:300), Alexa Fluor 594 chicken anti-rat lgG (Invitrogen, 479145, 1:300), Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, 2066710, 1:300), and Alexa Fluor 594 goat anti-mouse IgG (Invitrogen, 419361, 1:300).
The Quantitative real-time PCR is described in detail elsewhere. The primer pairs listed in Table 2 were used for the amplification of target genes.
Name Primer pairs (5′-3′) GAPDH F: AGTGCCAGCCTCGTCTCATA R: GATGGTGATGGGTTTCCCGT CD86 F: AAGCCCGTGTCCTTGATCTG R: AGACATGTGTAACCTGCACCAT CD163 F: AGCATGGCACAGGTCATTCA R: GGTCACAAAACTTCAACCGGA iNOS F: GAGACGCACAGGCAGAGGTTG R: AGCAGGCACACGCAATGATGG Arg-1 F: TGGACCCTGGGGAACACTAT R: GTAGCCGGGGTGAATACTGG IL-6 F: ACTTCCAGCCAGTTGCCTTCTTG R: TGGTCTGTTGTGGGTGGTATCCTC IL-10 F: CTGCTCTTACTGGCTGGAGTGAAG R: TGGGTCTGGCTGACTGGGAAG IL-12 F: CCTCAAGTTCTTCGTCCGCATCC R: CATTGGACTTCGGCAGAGGTCTTC IFN-γ F: CCTCAAGTTCTTCGTCCGCATCC R: CACCGACTCCTTTTCCGCTTCC TNF-α Forward: ATGGGCTCCCTCTCATCAGTTCC R: GCTCCTCCGCTTGGTGGTTTG TGF-β F: GACCGCAACAACGCAATCTATGAC R: CTGGCACTGCTTCCCGAATGTC
Table 2. Primer pairs used for amplification
The detailed method for western blot analysis is described elsewhere. The expression of iNOS, Arg-1 and IFN-γ protein was detected by western blotting. The primary antibodies used were: anti-iNOS (Servicebio, GB11119, 1:1,000), anti-Arginase-1 (Cell Signaling, #93668, 1:1,000), anti-GAPDH (ZSGB-Bio, TA-08, 1:2,000), and anti-IFN-γ (Bioss, bs-0480R, 1:500). The secondary antibodies were: goat anti-rabbit IgG (H+L) (Thermo Fisher, 31460, 1:5,000) and goat anti-mouse IgG (H+L) (Thermo Fisher, 31430, 1:5,000). The density of the protein bands was quantified using ImageJ software.
Experimental analysis was performed using SPSS software (version 20.0; Chicago, IL, USA). Data were expressed as mean ± standard deviation (SD) and were analyzed using GraphPad Prism version 6. The differences between groups were determined by one-way analysis of variance (ANOVA). P < 0.05, or P < 0.01, indicated a statistically significant difference.
Establishment of a Radiation-induced Abscopal Effect in Rat Model of Breast Cancer
Immunohistochemistry and Immunofluorescence
Quantitative Real-time PCR Analysis
Western Blot Analysis