Figure 6. Deletion of Muc4 suppresses the association of tumor cells with red and white blood cells in vitro and in vivo.

(A) Representative H&E stained lung lesions from Muc4^wt/NDL and Muc4^ko/NDL mice, illustrating the elevated abundance of blood cells (closed arrowheads) in Muc4^wt/NDL emboli. Immunodetection of proteins specific to macrophages (F4/80) and platelets, megakaryocytes and hematopoietic progenitors (CD41/Integrin alpha 2b) indicated the majority of associated blood cells to be CD41 positive. Phospho-histone H3 (pH3) staining revealed Muc4^wt/NDL lung lesions were more proliferative than Muc4^ko/NDL lesions. Scale bar in all images = 100μm. Images are representative of at least 6 biological replicates. (B) Quantification of tumor cell-blood cell associations (proximity of < 5μm) using bright field microscopy of live cells revealed a reduced number of associated blood cells at either 1.5hrs or 24hrs after the initiation of co-culture conditions. Pooled NDL cells (n=4 biological sources per line) were used for these experiments and data are presented as averages of three independent experiments ± SD. Scale bar = 50μm. *, P < 0.05. (C) Similar associations were noted for Muc4^wt/NDL cells in vivo, where the abundance of closely associated blood cells appears visibly enhanced by the presence of Muc4. Pooled Muc4^wt/NDL or Muc4^ko/NDL cells (n=4 biological sources per line) were instilled into the lateral tail vein of female mice (n=4 per line) and whole blood was collected by cardiac puncture 3d later and immediately fixed in 4% paraformaldehyde. Nuclei were stained with DAPI and individual cell clusters imaged by fluorescence and bright field microscopy. Scale bar = 200μm. (D) Muc4-mediated tumor cell association with blood cells promotes tumor cell viability in suspension. Primary Muc4^wt/NDL and Muc4^ko/NDL tumor cells (n=3 per line) were labeled with MitoFluor green, and cultured in suspension for 24hrs after admixing with basal media, freshly harvested blood cells in basal media, or cell free-plasma (CFP) in basal media harvested from Muc4^wt or Muc4^ko mice. Hoechst 33342 was used as a nuclear stain to discern platelets from immune and tumor cells, and MitoFluor green was used to distinguish tumor cells from immune cells. Percent cytotoxicity (mean ± SD) was determined within tumor sphere aggregates by positive staining for ethidium homodimer. Student’s t-test revealed blood cells significantly suppress the death of Muc4^wt/NDL but not Muc4^ko/NDL cells. The origin of the blood cells (Muc4^wt or Muc4^ko) did not affect outcomes. Images are presented as final merged composites. Unmerged images may be found in Supplemental Figure 4B. Scale bar = 200μm. *, P < 0.05; ***, P < 0.001.