Modulation of tumor microenvironment by targeting cancer associated fibroblasts in dendritic cell based immunotherapy


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Sheefa Mirza1,Clement Penny1,Nayan Jain2,Rakesh Rawal2
1University of Witwatersrand,2Gujarat University

Abstract

Background

Cancer associated fibroblasts (CAFs), main component of TME, have an important role in generating immunosuppresive TME by modulating recruitment and functions of tumor-associated immune cells by secreting various growth hormones, miRNAs and cytokines. Curcumin is known to modulate numerous target proteins including transcription factors, receptors, kinases, cytokines, enzymes and growth factors. Thus, aim of the study was to evaluate the effect of miRNAs and cytokines released by lung cancer patients’ derived CAFs and to assess immunomodulatory potential of curcumin on DC maturation by targeting these CAFs through modulating their TME.

Method

CAFs were cultured and characterized using CAF-specific markers. rGM-CSF and rIL-4 were used to generate immature DCs (imDCs).Further, imDCs were cultured in conditioned media derived from CAFs (CAFs-CM) as well as NFs (Normal Fibroblasts) to evaluate the effect of CAFs on DC maturation. mDCs were characterized by qRT-PCR using CD80, CD83, CD86 and CTLA4. Moreover, miR-221, miR-222, miR-155, miR-142-3p and miR-146a were assessed to evaluate their epigenetic regulation on DC maturation. Cytokine profiling of CAFs-CM as well as CAFs-CM treated with curcumin was conducted.

Results

α-SMA+Vimentin+ cells were considered as CAFs. Significant upregulation of CD80, CD83 and CD86 was observed when cultured in NFs-CM while a remarkable downregulation of these markers was found when cultured in CAFs-CM. CTLA-4 was down regulated in NFs-CM as compared to CAFs-CM, suggesting the role of CAFs in generation of regulatory DCs. Amongst all miRNAs, miR-146a was shown to be up regulated dramatically in CAF-DCs (DCs cultured in CAFs-CM) as well as in CAFs-CM, suggesting the immunosuppressive role of miR-146a. Further, an increased expression of miR-146a was positively correlated with increased expression of anti-inflammatory cytokines like IL-6, IL-10, TGF-β and decreased expression of TNF-α (pro-inflammatory) in CM derived from CAF-DCs. Moreover, curcumin had the potential to convert regulatory DCs facilitated by CAFs into mDCs, which were characterized by high expression of co-stimulatory molecules, low expression of CTLA4, lower levels of immune suppressive cytokines production, lower levels of miR-146a.

Conclusion

These findings provide insight into understanding the immunomodulatory role of curcumin in targeting CAFs and modulating the tumor microenvironment, thus enhancing antitumor immune response in DC based therapy.