Fig 1.
Schematic overview of the methodology used in this work.
Monocytes were isolated from the peripheral blood of healthy blood donors, and cultured, for seven days, with M-CSF to allow their differentiation into macrophages. On day 11, RKO or SW1463 cancer cells were cultured in transwell inserts of 1 μm pore size, on top of macrophages, and the whole set was then irradiated with 2 Gy/fraction/day, for 5 days. Conditioned medium (CM) of irradiated co-cultures, as well as protein and RNA from individual cell populations, were collected 6 h after the last ionizing radiation dose, and compared with the respective controls.
Fig 2.
Expression of total and cleaved forms of PARP and caspase-3 in RKO and SW1463 cancer cells.
A) RKO and B) SW1463 cancer cells were cultured alone (-) or in the presence of macrophages (ccMac), with (IR or ccIR, 5 x 2 Gy) or without (Ctr or ccCtr) radiation exposure. Expression levels of total and cleaved forms of PARP and caspase-3 were evaluated 6 h after irradiation by western blot analysis.
Fig 3.
Expression of survival- and metabolism-related targets in RKO and SW1463 cancer cells.
Both RKO and SW1463 cancer cells were cultured alone (-) or in the presence of macrophages (ccMac), with (IR, 5 x 2 Gy) or without (Ctr) radiation exposure. The mRNA expression levels of A) two survival-related genes, BCL2L1 and MCL1, which encode the anti-apoptotic proteins Bcl-xL and Mcl-1, respectively, and B) of two metabolism-related genes, SLC2A1 and LDHA, which encode the glucose transporter type 1 and the lactate dehydrogenase, respectively, were evaluated in cancer cells, 6 h after irradiation. Graphs result from the relative mRNA quantification in cancer cells cultured with macrophages from distinct donors (n = 4 per each cell line), evaluated in four independent experiments. * P < 0.05, ** P < 0.01.
Fig 4.
The mRNA expression of pro-inflammatory markers in irradiated macrophages, cultured with RKO or SW1463 cells.
Macrophages were cultured alone (-) or in the presence of RKO or SW1463 cancer cells (ccRKO or ccSW), with (IR, 5 x 2 Gy) or without (Ctr) radiation exposure. The mRNA expression of a panel of pro-inflammatory macrophage markers (TNF, IL6, CCL2, CCR7, IL1B, CXCL8, CD80 and CCL5) was evaluated 6 h after irradiation. Graphs result from the relative mRNA quantification in macrophages cultured with RKO or SW1463 (n = 4 per each cell line), evaluated in four independent experiments. For simplicity, SW1463 cells were indicated as “SW”. * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 5.
The mRNA expression of anti-inflammatory markers in irradiated macrophages, cultured with RKO or SW1463 cells.
Macrophages were cultured alone (-) or in the presence of RKO or SW1463 cancer cells (ccRKO or ccSW), with (IR, 5 x 2 Gy) or without (Ctr) radiation exposure. The mRNA expression of a panel of anti-inflammatory macrophage markers (IL10, CD163, CCL18 and VCAN) was evaluated 6 h after irradiation. Graphs result from the relative mRNA quantification in macrophages cultured with RKO or SW1463 (n = 4 per each cell line), evaluated in four independent experiments. For simplicity, SW1463 cells were indicated as “SW”. * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 6.
Effect of conditioned medium from irradiated co-cultures in the invasion, migration and angiogenesis of non-irradiated cells.
The ability of conditioned medium (CM), from irradiated (IR, 5 x 2 Gy) and non-irradiated (Ctr) co-cultures (cc) of macrophages with RKO or SW1463, to promote cancer cell migration and invasion (n = 4 per each cell line), as well as to induce angiogenesis, was evaluated. A) Non-irradiated RKO and SW1463 cells were seeded on Matrigel-based transwells and stimulated with CM from irradiated and non-irradiated co-cultures. After 24 h, the number of invasive cells was counted. B) Both non-irradiated RKO or SW1463 were plated until confluence on both chambers of Ibidi culture-inserts for migration assay. After wound formation, cancer cells were stimulated with CM from irradiated and non-irradiated co-cultures and migrated area was quantified upon 48 h. C) Concentrated CM from irradiated or non-irradiated co-cultures was inoculated in rings, on the top of the chick embryo chorioallantoic membrane (CAM), for 72 h. Analysis of CM-induced angiogenesis was performed through quantification of the number of new vessels in control and experimental conditions.
Fig 7.
Intricate macrophage-cancer cell communication upon irradiation.
I) In the presence of macrophages, radiosensitive RKO cell increased cleaved PARP and cleaved caspase-3 expression in response to radiation, while radioresistant SW1463 reduced the expression of these targets. The high BCL2L1, MCL1 and SLC2A1 levels observed in SW1463 cells upon co-culture with macrophages, together with the increased expression of BCL2L1 in irradiated SW1463 upon co-culture with macrophages, may contribute to macrophage-induced SW1463 enhanced radioresistance. II) In the presence of either RKO or SW14363 cancer cells, irradiated macrophages exhibit higher levels of pro-inflammatory TNF, IL6, CCL2 and CCR7, but also of anti-inflammatory CCL18, being differences in other targets dependent on the nature of the cancer cells with which macrophages were cultured. Thus, in the presence of RKO cells irradiated macrophages exhibit an increase of pro-inflammatory IL1B, while SW1463 cells promote higher pro-inflammatory CXCL8 and CD80, but also anti-inflammatory VCAN and IL10 levels. III) Conditioned medium (CM) from macrophage-RKO irradiated co-culture induced increased invasion and migration of non-irradiated RKO cells. Abbreviations: cc–co-cultures; IR–irradiated.