The impact of HSD11B1-mediated activation of glucocorticoids in the pathogenesis and resistance to immunotherapy in malignant melanoma
Therefore, a syngeneic melanoma mouse model was used to characterize the tumorigenic potential of HSD11B1 in vivo, specifically to elucidate how HSD11B1 inhibition in combination with αPD-1 blockade (combination therapy) affects tumor growth and immune infiltration compared to αPD-1 monotherapy. To do so, comprehensive immune cell flow cytometry panels were established, and immunofluorescence used to confirm tumoral immune cell infiltration. Additionally, cytokine profiles were analyzed. A novel 3D technique, light sheet fluorescence microscopy was applied to spatially visualize and quantify the distinct immune cell population under combination therapy versus αPD-1 monotherapy. To mimic melanoma tumor heterogeneity, murine melanoma cell lines ectopically expressing HSD11B1-recurrent mutations were generated and cells were labeled by using a single cell tracking barcoding technology. Upon in vivo transplantation, mixed cell populations were quantified individually regarding their proliferation capabilities under combination therapy. Finally, to validate HSD11B1 as potential predictive biomarker, tumor tissues from melanoma patients collected before and under PD-1 therapy were used for HSD11B1 immunostaining.
Combination therapy significantly reduced melanoma tumors and increased the recruitment of immune cells, specifically CD45+ cells, macrophages and dendritic cells. Interestingly, combination therapy efficacy was dependent on the activation of CD8+ T cell. Mechanistically, HSD11B1 inhibition in combination with PD-1 blockade augmented the production of IFN-γ by infiltrating CD8+ T cells. Upon CD8+ T cell depletion with antibodies, combination therapy was largely inefficient. Furthermore, HSD11B1 inhibition promoted long-lasting tumor control in combination therapy-treated melanoma mice, but also with the addition of HSD11B1 inhibitor in melanoma mice initially treated with αPD-1 monotherapy. Moreover, combination therapy reduced the expression of cytokines associated to tumor proliferation and metastasis compared to αPD-1 monotherapy. Additionally, HSD11B1-recurrent mutations in melanoma did not affect therapy efficacy. Finally, in melanoma patient tumor samples, high expression of HSD11B1 was significantly associated with poor response to ICIs.
Overall, these results suggest a new therapeutical strategy to improve PD-1 monotherapy efficacy and to overcome therapy resistance for the treatment of malignant melanoma, boosting immune responses. As inflammation is induced by HSD11B1 inhibition, this work also delineated potential limitations emphasizing the need of further studies regarding duration of therapy and careful patient stratification with baseline CD8+ T cells detection.
Es wurde ein syngenes Melanom-Mausmodell verwendet, um das tumorerzeugende Potenzial von HSD11B1 in vivo zu charakterisieren und insbesondere zu klären, wie die HSD11B1 Inhibition in Kombination mit einer αPD-1 Blockade (Kombinationstherapie) das Tumorwachstum und die Immunzellinfiltration im Vergleich zur αPD-1 Monotherapie beeinflusst. Dafür wurden umfassende Immunzell-Durchflusszytometrie-Panels etabliert und die Immunfluoreszenz verwendet, um die Immunzellinfiltration in Tumoren zu bestätigen. Zusätzlich wurden Zytokin Profile analysiert. Eine neue 3D-Technik, die Lichtscheibenfluoreszenzmikroskopie, wurde benutzt um die Immunzellpopulationen unter Kombinationstherapie im Vergleich zur αPD-1 Monotherapie räumlich darzustellen und zu quantifizieren. Um die Melanomheterogenität nachzuahmen, wurden murine Melanomzelllinien, welche ektopisch HSD11B1 wiederkehrende Mutationen exprimieren, generiert und die Zellen mit der Barcoding-Technologie zur Einzelzellverfolgung markiert. Nach der in vivo Transplantation wurden die gemischten Zellpopulationen einzeln hinsichtlich ihrer Proliferationsfähigkeit unter der Kombinationstherapie quantifiziert. Um schließlich HSD11B1 als einen potenziellen prädiktiven Biomarker zu validieren, wurde Tumorgewebe von Melanompatienten, das vor und unter einer PD-1 Therapie gesammelt wurde, für HSD11B1 Immunfärbungen verwendet.
Die Kombinationstherapie reduzierte Melanomtumore signifikant und verstärkte die Rekrutierung von Immunzellen, insbesondere von CD45+-Zellen, Makrophagen und dendritischen Zellen. Interessanterweise war die Effektivität der Kombinationstherapie von der