Figure 6.

PGE₂/EP2 axis is the driver of biopsy-induced M2 polarization, EMT, and systemic dissemination

(A) RT-qPCR analysis of Arg1 mRNA expression in the primary Mφ co-treated with PGE₂ and antagonists for EP1 (10 nM, ONO8711), EP2 (1 μM, PF04418948), EP3 (10 nM, L-798106), or EP4 (1 μM, GW-627368X). Relative expression was compared between five treatment groups using one-way ANOVA, relative to PGE₂ treatment (n = 4–8). The data are shown as standard boxplots.

(B) Mφ treated with EP receptor agonist for EP1 + EP3 (1 μM, 17PT PGE₂), EP2 (150 nM, CP544326), or EP4 (100 nM, ONO-4819) for Arg1 mRNA expression was compared using one-way ANOVA, relative to control (n = 4). The data are shown as standard boxplots.

(C) Arg1 mRNA expression in Ep2^+/+ Mφ and Ep2^−/− Mφ treated with or without PGE₂ (n = 4) was compared using two-way ANOVA, relative to control. The data are shown as standard boxplots.

(D) Ep2 mRNA expression in Mφ treated with or without PGE₂ under hypoxia (n = 4) was compared using Student’s t test. The data are shown as standard boxplots.

(E) IF images of VEGF and TGF-β1 of Ep2^+/+ Mφ and Ep2^−/− Mφ treated with or without PGE₂ under hypoxia (n = 4).

(F) Schematic diagram of experimental flow of Mφ adoptive transfer. Py230 tumor-bearing recipient mice were treated with clodronate once for Mφ depletion and then received adoptive transfer of Mφ isolated from Ep2^+/+ or Ep2^−/− donor mice, after which they received biopsy or were left unbiopsied. Tumors were isolated 15 days after the biopsy.

(G) Immunoprofiling of Mφ isolated from Py230 tumors of Ep2^+/+ Mφ or Ep2^−/− Mφ recipients (n = 7). The data are shown as standard boxplots; p values were calculated using two-way ANOVA, relative to the unbiopsied control.

(H) Representative images of two distinct areas adjacent to or distant from biopsy wound in 15-day post-biopsy Py230 tumors, immunofluorescently stained for CD31 (pink), CK (green), and vimentin (red), and counterstained with DAPI (blue). Graph summarizes the percentage of vimentin⁺/CK⁺ cells and CD31⁺ blood vessel count, normalized by nucleus count per field of view at the final magnification of 40× (n = 5). The data are shown as mean ± SEM; p values were calculated using two-way ANOVA, relative to distant.

(I) Graph depicting the number of metastatic Py230^mCherry cells in the lung of Ep2^+/+ Mφ or Ep2^−/− Mφ recipient mice that were biopsied or unbiopsied (n = 10–14). Data were plotted as means ± SEM; p values were calculated using two-way ANOVA, relative to the unbiopsied control.