Background

Emerging data suggest that patients with enzalutamide-treated prostate cancer with increased programmed death-ligand 1 (PD-L1) expression may benefit from anti-PD-L1 treatment. Unfortunately, the Phase III IMbassador250 clinical trial revealed that the combination of atezolizumab (a PD-L1 inhibitor) and enzalutamide failed to extend overall survival in patients with castration-resistant prostate cancer (CRPC). However, the mechanisms underlying treatment failure remain unknown.

Methods

Human CRPC C4-2B cells and murine Myc-CaP cells were chronically exposed to increasing concentrations of enzalutamide and the cells resistant to enzalutamide were referred to as C4-2B MDVR and Myc-CaP MDVR, respectively. The mechanisms of action in drug-resistant prostate cancer cells were determined using RNA sequencing analyses, RNA interference, real-time PCR, western blotting, and co-culturing technologies. Myc-CaP and Myc-CaP MDVR tumors were established in syngeneic FVB mice, and tumor-infiltrating leukocytes were isolated after enzalutamide treatment. The stained immune cells were determined by flow cytometry, and the data were analyzed using FlowJo.

Results

Immune-related signaling pathways (interferon alpha/gamma response, inflammatory response, and cell chemotaxis) were suppressed in human enzalutamide-resistant prostate cancer cells. PD-L1 was overexpressed and negatively regulated by androgen receptor signaling in resistant cells and patient with CRPC cohorts. Enzalutamide treatment decreased CD8⁺ T-cell numbers but increased monocytic myeloid-derived suppressor cell (M-MDSC) populations and PD-L1 expression within murine Myc-CaP tumors. Similarly, chemotaxis and immune response-regulating signaling pathways were suppressed, and PD-L1 expression was also increased using enzalutamide-resistant Myc-CaP MDVR cells. Notably, MDSC populations were significantly increased in Myc-CaP MDVR orthotopic tumors compared with those in Myc-CaP parental tumors. Co-culturing bone marrow cells with Myc-CaP MDVR cells significantly promoted MDSC differentiation and shifted towards M2 macrophage skewing.

Conclusions

Our study suggests that immunosuppressive signaling can be promoted directly by enzalutamide-resistant prostate cancer cells and may be a potential means by which the efficacy of immune checkpoint inhibitors in enzalutamide-resistant prostate cancer is diminished.

Introduction

Immune checkpoint inhibitors (ICIs) only benefit a subset of cancer patients, underlining the need for predictive biomarkers for patient selection. Given the limitations of tumor tissue availability, flow cytometry of peripheral blood mononuclear cells (PBMCs) is considered a noninvasive method for immune monitoring. This study explores the use of spectrum flow cytometry, which allows a more comprehensive analysis of a greater number of markers using fewer immune cells, to identify potential blood immune biomarkers and monitor ICI treatment in non-small-cell lung cancer (NSCLC) patients.

Methods

PBMCs were collected from 14 non-small-cell lung cancer (NSCLC) patients before and after ICI treatment and 4 healthy human donors. Using spectrum flow cytometry, 24 immune cell markers were simultaneously monitored using only 1 million PBMCs. The results were also compared with those from clinical flow cytometry and bulk RNA sequencing analysis.

Results

Our findings showed that the measurement of CD4+ and CD8+ T cells by spectrum flow cytometry matched well with those by clinical flow cytometry (Pearson R ranging from 0.75 to 0.95) and bulk RNA sequencing analysis (R=0.80, P=1.3 x 10-4). A lower frequency of CD4+ central memory cells before treatment was associated with a longer median progression-free survival (PFS) [Not reached (NR) vs. 5 months; hazard ratio (HR)=8.1, 95% confidence interval (CI) 1.5-42, P=0.01]. A higher frequency of CD4-CD8- double-negative (DN) T cells was associated with a longer PFS (NR vs. 4.45 months; HR=11.1, 95% CI 2.2-55.0, P=0.003). ICIs significantly changed the frequency of cytotoxic CD8+PD1+ T cells, DN T cells, CD16+CD56dim and CD16+CD56- natural killer (NK) cells, and CD14+HLDRhigh and CD11c+HLADR + monocytes. Of these immune cell subtypes, an increase in the frequency of CD16+CD56dim NK cells and CD14+HLADRhigh monocytes after treatment compared to before treatment were associated with a longer PFS (NR vs. 5 months, HR=5.4, 95% CI 1.1-25.7, P=0.03; 7.8 vs. 3.8 months, HR=5.7, 95% CI 169 1.0-31.7, P=0.04), respectively.

Conclusion

Our preliminary findings suggest that the use of multicolor spectrum flow cytometry helps identify potential blood immune biomarkers for ICI treatment, which warrants further validation.

In leukemia, a distinct subpopulation of cancer-initiating cells called leukemia stem cells (LSCs) is believed to drive population expansion and tumor growth. Failing to eliminate LSCs may result in disease relapse regardless of the amount of non-LSCs destroyed. The first step in targeting and eliminating LSCs is to identify and characterize them. Acute precursor B lymphoblastic leukemia (B-ALL) cells derived from patients were incubated with fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose (NBDG) and sorted based on NBDG uptake. Cell subpopulations defined by glucose uptake were then serially transplanted into mice and evaluated for leukemia initiating capacity. Gene expression profiles of these cells were characterized using RNA-Sequencing (RNA-Seq). A distinct population of NBDG-low cells was identified in patient B-ALL samples. These cells are a small population (1.92% of the entire leukemia population), have lower HLA expression, and are smaller in size (4.0 to 7.0 μm) than the rest of the leukemia population. All mice transplanted with NBDG-low cells developed leukemia between 5 and 14 weeks, while those transplanted with NBDG-high cells did not develop leukemia (p ≤ 0.0001-0.002). Serial transplantation of the NBDG-low mouse model resulted in successful leukemia development. NBDG-medium (NBDG-med) populations also developed leukemia. Interestingly, comprehensive molecular characterization of NBDG-low and NBDG-med cells from patient-derived xenograft (PDX) models using RNA-Seq revealed a distinct profile of 2,162 differentially-expressed transcripts (DETs) (p<0.05) with 70.6% down-regulated in NBDG-low cells. Hierarchical clustering of DETs showed distinct segregation of NBDG-low from NBDG-med and NBDG-high groups with marked transcription expression alterations in the NBDG-low group consistent with cancer survival. In conclusion, A unique subpopulation of cells with low glucose uptake (NBDG-low) in B-ALL was discovered. These cells, despite their quiescence characteristics, once transplanted in mice, showed potent leukemia initiating capacity. Although NBDG-med cells also initiated leukemia, gene expression profiling revealed a distinct signature that clearly distinguishes NBDG-low cells from NBDG-med and the rest of the leukemia populations. These results suggest that NBDG-low cells may represent quiescent LSCs. These cells can be activated in the appropriate environment in vivo, showing leukemia initiating capacity. Our study provides insight into the biologic mechanisms of B-ALL initiation and survival.