Figure 4.

Protracted activation of COX-2 in infiltrating BMDCs in biopsy wounds promotes EMT and systemic dissemination of BC

(A) Representative images of COX-2 staining of wound stroma cells in the biopsy cavity in 50-day post-biopsy stage I clinical BC and 15-day post-biopsy Py230 tumor. Insets indicate the distant stroma from the biopsy wound.

(B) Schematic diagram of experimental flow of in vivo Cox-2 promoter assay. Six weeks following the adoptive transfer of BMDCs isolated from Cox-2^luc/luc mice into lethally irradiated recipient mice, Py230 cells were orthotopically injected. Tumors were biopsied or left unbiopsied. Bioluminescence was detected immediately after intravenous injection of D-luciferin using an IVIS in vivo imaging system.

(C) Cox-2 promoter activity at indicated times in individual mice that received biopsy (red) or left unbiopsied (blue). The graph depicts the fold change of total flux in the region of interest, relative to day 0 (before biopsy). Images depict bioluminescence on the tumor of individual mice over the experimental period. Data were analyzed in a linear mixed-effects model with random effects of mice and fixed effects of time after biopsy (n = 3).

(D) PGE₂ quantification of Py230 tumors at the indicated times after biopsy using ELISA. Data were normalized by tumor weight and expressed as pg/mg tumor (n = 5). A simple linear regression was used to model the effect of time after biopsy on the levels of PGE_2, relative to day 0. The curve was fitted using an exponential function.

(E) Western blot analysis of whole tumor lysate of Py230 tumors for COX-1, COX-2, and mPGES2 expression. The graph depicts the relative expression normalized by GAPDH (n = 4). The data are shown as mean ± SEM; p values were calculated using the Wilcoxon test, relative to the unbiopsied control.

(F) Schematic diagram of experimental flow of Cox-2^luc/luc or Cox-2^+/+ BMDC adoptive transfer. Six weeks following the adoptive transfer of BMDCs isolated from Cox-2^luc/luc or Cox-2^+/+ donor mice into lethally irradiated B6 recipient mice, Py230 cells were orthotopically injected. Tumors were biopsied or left unbiopsied and collected 15 days later.

(G) Immunoprofiling of Mφ phenotype of biopsied or unbiopsied Py230 tumors in Cox-2^luc/luc recipients (n = 6). The data are shown as standard boxplots; p values were calculated using Wilcoxon test.

(H) Graph depicting the number of metastatic Py230^mCherry cells in the lungs of Cox-2^luc/luc and Cox-2^+/+ recipients that were unbiopsied or biopsied (n = 8–9). Data were plotted as mean ± SEM and analyzed by two-way ANOVA, relative to unbiopsied control.

(I) Representative images of 15-day post-biopsy Py230 tumors adjacent to or distant from the biopsy wound, from Cox-2^luc/luc and Cox-2^+/+ recipients, immunofluorescently stained for CK (green) and vimentin (red) and counterstained with DAPI (blue). BW, biopsy wound. Graph on right summarizes the percentage of vimentin⁺/CK⁺ cells 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.