PMC

Control mice were sacrifice on day 6, 8 and 12. Irradiated mice were sacrificed on day 6, 8, 12, 15 and 17. Tumors, blood, spleens, lungs and lymph nodes were collected for FACS analysis for MDSC and TAM population. N=4 for each time point.

Subcutaneous RM-1 tumors were collected, processed to single cell suspension and assayed by FACS and immunohistochemistry for CD11b⁺Gr-1⁺ MDSCs and CD11b⁺F4/80⁺ TAMs. A) Growth curve of RM-1 tumors with or without irradiation. B) FACS plots and quantification of TAMs in tumor. C) FACS plots and quantification for MDSCs in tumor. D) Representative F4/80 staining of RM-1 tumors from control and irradiation-treated mice. E) Effect of irradiation on the two subsets of MDSCs: MO-MDSC and PMN-MDSC. N=4 for each group.

A) Tumor irradiation activates ABL1, which translocates to the nucleus, binding to the promoter region of CSF1 and up-regulates its gene expression. Additional TIMs are recruited to tumor sites due to the increase in CSF1 and they can thus promote tumor growth. B) When tumor-bearing mice were treated with a small molecule CSF1R kinase inhibitor, CSF1/CSF1R signaling is inhibited resulting in decreased infiltration of TIMs, reducing their tumor growth promoting influences.

A) Growth curve of subcutaneous RM-1 tumors treated with RT (3Gy×5 days), PLX3397 (in food chow) or combination as indicated. Tumors were measured daily by caliper. B–C) FACS analysis of CD11b⁺F4/80⁺ macrophages and CD11b⁺Gr-1⁺ MDSCs in tumor, collected at end points (tumor n=6 × for each cohort). D–E) FACS analysis of CD11b⁺F4/80⁺ macrophages and CD11b⁺Gr-1⁺ MDSCs in spleen, at the same termination time as above. F) FACS analysis for MDSC subsets, MO-MDSC and PMN-MDSC with single or combination treatment. G) Representative immunohistochemsitry staining for F4/80 on tumor sections with single or combination treatment. H–J) RT-PCR analysis of mRNA extracted from tumors for CSF1, MMP9 and Arg1.

A) MycCaP cells were plated overnight and irradiated with 3Gy. Cells were collected 24 hrs after, and assayed by RT-PCR for factors known to recruit myeloid cells: CSF1, CCL2, SDF1 and CCL5. CSF1 is shown to have the highest expression and most significant increase. B) Conditioned media from Myc-CaP cells 48hrs after irradiation were collected, concentrated by centrifugation, normalized and analyzed by SDS-PAGE. Secreted CSF1 was detected at 50kDa. Relative expression level quantified by ImageJ was shown below the blot. C) Tumors collected as shown in were assayed by RT-PCR for CSF1 mRNA expression. D) Serum samples from prostate cancer patients pre- and post-radiotherapy were analyzed by ELISA for CSF1. E) RM-1 cells and macrophage cell line RAW264.7 were co-cultured overnight and irradiated with 3Gy. Cells were collected 24hrs and analyzed by RT-PCR for CSF1 mRNA. F) RM-1 cells and BMDM were co-cultured for 4hrs and irradiated with 3Gy. Cells were collected 24hrs and analyzed by RT-PCR for CSF1 mRNA. G) RAW264.7 macrophage migration assay was performed by using conditioned media in lower chamber, collected as in panel B. GW2580 (1μM) was added to the top chamber. Quantification of 9 fields was summarized in panel F. Representative images of migrated cells were shown in G.

A) RAW264.7 macrophages were seeded in 8-µm transwell inserts, and tumor conditioned media (collected 48hrs after 3Gy irradiation) was placed in the bottom. Cells were allowed to migrate toward bottom for 6 hrs. Then cells were fixed and stained with DAPI. Representative images of migrated cells are shown and they were quantified using ImageJ software (n=3). B) Effect of irradiation on bone marrow derived macrophages (BMDM). Bone marrows were collected and induced to macrophages by CSF1 (10ng/ml) for 6 days. Cells were counted, seeded and subjected to 3Gy irradiation. Cells were collected 24hrs later, and RT-PCR was performed to detect RNA for the protumorigenic and inflammatory genes noted. C) BMDMs as prepared above were cultured in 50% tumor conditioned media + 50% complete DMEM for 24hrs. Cells were collected and assayed by RT-PCR for the genetic markers noted. D) Tumors collected as shown in were analyzed by FACS for MHCII expression on CD11b⁺F480⁺ macrophages. An increase in MHCII low-expressing macrophage population was observed. (* indicates significant changes with P< 0.05)

A) CSF1 mRNA expression in Myc-CaP cells 0, 1, 2, 4, 6, 8, and 24hrs after irradiation (3Gy). B) ABL1 is cleaved by irradiation in vitro. 4×10⁵ Myc-CaP cells were plated in 6-well plate overnight and irradiated with 3Gy the next day. Cells were collected at 1, 2, 4, and 8 hrs after irradiation. Cells lysates were normalized and assayed by SDS-PAGE. Western blot was probed with ABL1 (K-12) antibody for both full length and cleaved ABL1. Two cleavage products, 60kD and 75kD, were detected. C) Confocal images of Myc-CaP cells at 1 and 4hrs after irradiation. Green: ABL1; red: actin; white: nucleus (DAPI). D–E) Myc-CaP cells 0, 1, 2, 4 hrs after irradiation were fixed in 3% PFA and processed for ChIP assay using c-Abl antibody (D) and RNA polymerase II antibody (E) as described in Material and Methods section. Rabbit IgG was used as negative control. F) Myc-CaP cells were irradiated with 3Gy and STI-571 (5μM) was added right after. Cells were collected 24hrs later and analyzed by RT-PCR for CSF1 mRNA expression. G) Migration assay using conditioned media collected as in panel F. GW2580 was added to the top chamber to examine the additive effects between STI-571 and GW2580. H) CSF1-mRNA in human prostate cancer cells after ABL1 directed RNAi. CWR22Rv1 cells were transfected with ABL1 siRNA and negative control (NC: non-specific siRNA). Cells were irradiated 30hrs after siRNA treatment and collected 24hrs after irradiation. Expression level of ABL1 was reduced to 30% of control after specific siRNA treatment (right panel). CSF1 mRNA was analyzed by RT-PCR with and without irradiation.