Abstract
In the present study, we aimed to investigate the influence of lactate shuttling on the functional polarization and spatial distribution of transitional cell carcinoma of the bladder (TCCB) cells and macrophages. We designed a microfluidic coculture chip for real-time integrative assays. The effect of lactate shuttling on the re-education of macrophages by TCCB cells was explored by measuring the levels of NO using a total NO assay kit and by evaluating the protein expression of iNOS, p-NFkB-p65, Arg-1 and HIF-1α via cell immunofluorescence and western blotting. Additionally, we examined TCCB cell viability using acridine orange/ethidium bromide (AO/EB) and MitoTracker staining. Moreover, the concentration distributions of lactate and large signaling proteins in the culture chambers were measured using 4',6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate-dextran (FITC-dextran). Furthermore, the recruitment of macrophages and the influence of macrophages on BC metastasis were observed via light microscopy. We confirmed that TCCB cells reprogrammed macrophages into an M2 phenotype. Moreover, lactate inhibited M1 polarization and induced M2 polarization of macrophages, but blockade of cancer cell-macrophage lactate flux significantly inhibited the re-education of macrophages by TCCB cells. In addition, lactate diffused faster and deeper than large signaling proteins in the microfluidic tumor microenvironment. Furthermore, lactate alone induced the migration of macrophages, and M1, but not M2, macrophages reduced the motility of TCCB cells. TCCB cells reprogrammed macrophages into an M2 phenotype in a manner that depended on cancer cell-TAM lactate flux. Furthermore, the lactate shuttle may be a determinant of the density of TAMs in tumor tissue.
Highlights
Bladder cancer (BC) is one of the most common malignancies worldwide, with approximately 73,000 newly diagnosed cases and 15,210 cancer-related deaths in 2013 in the USA [1]
Lactate alone induced the migration of macrophages, and M1, but not M2, macrophages reduced the motility of TCCB cells
TCCB cells reprogrammed macrophages into an M2 phenotype in a manner that depended on cancer cell-tumor-associated macrophages (TAMs) lactate flux
Summary
Bladder cancer (BC) is one of the most common malignancies worldwide, with approximately 73,000 newly diagnosed cases and 15,210 cancer-related deaths in 2013 in the USA [1] Cancer immunotherapies such as intravesical Bacillus Calmette-Guerin (BCG) therapy have prevalently been utilized for BC treatment [2]. TAMs are remarkably plastic cellular components in the tumor microenvironment These cells exhibit two oppositely programmed phenotypes: a classically activated phenotype (M1), which is observed in early stage tumors, and an alternatively activated phenotype (M2), which is observed in tumors that have progressed [7]. According to their opposing roles in cancer, two different cancer cell-macrophage interaction patterns correspond to an inflammatory or an anti-inflammatory pathway. We aimed to investigate the influence of lactate shuttling on the functional polarization and spatial distribution of transitional cell carcinoma of the bladder (TCCB) cells and macrophages
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