EURURO Vol. 71 No. 5

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Ingenuity Pathway Analysis tool for assignment of altered
canonical pathways and identification of upstream regulators.
According to analysis of 617 upregulated and 906 down-
regulated gene transcripts, AR-driven CRPC metastases had
higher metabolic activity for biosynthesis of cholesterol,
pyrimidines, and spermine and degradation of fatty acids
and amino acids when compared to non–AR-driven samples
. Among the downregulated canonical pathways in AR-
driven metastases, the cellular immune response was the most
obvious . Upstreamregulators predicted as responsible
for the differential expression observed between AR-driven and
non–AR-driven CRPC bone metastases are listed in Supple-
mentary Table 4. AR, SPDEF, and FOXA1 were among the top
activated genes that also showed increased mRNA levels in AR-
driven metastases, while several immune regulating genes
such as TGFB1, INFG, and other cytokines were predicted to be
inhibited, and some (CCL5, ETV5, PLAUR, and IFNAR2) also
showed reduced mRNA levels (Supplementary Table 4).
The predicted difference in cellular immune response
was verified by immunohistochemical analysis of CD3+
[3_TD$DIFF]
and CD68+ cells in CRPC bone metastases in FFPE
tissue available, which revealed a significantly higher
volume density of CD68+
[5_TD$DIFF] monocytes and borderline higher
frequency of CD3+ infiltrating lymphocytes in non–
AR-driven compared to AR-driven bone metastases
A–D). Gene expression data indicated higher
levels of CD3+ T cells, and specifically of CD8 + effector
T cells and CD4 + helper T cells in non–AR-driven compared
to AR-driven metastases (Supplementary Fig. 1A–C). This
was accompanied by increased levels of the inhibitory T
cell receptors CTLA4 and PDCD1 and lower levels of the
proinflammatory Th1 transcription factor TBX21 (Supple-
mentary Fig. 1D–F), while levels of the stimulatory T-cell
receptors ICOS and CD28 and the anti-inflammatory
Th2 transcription factor GATA did not differ between
non–AR-driven and AR-driven metastases (data not
shown). We did not detect mRNA for the regulatory T
cell (Treg) transcription factor FOXP3. Non–AR-driven
cases had higher levels of CD68, CD163, and S100A9 mRNA
(Supplementary Fig. 1G–I) but not NOS2 mRNA (data not
shown), indicating metastasis infiltration of tumor-pro-
moting M2 macrophages and myeloid-derived suppressor
cells (MDSCs).
The downregulated antigen presentation observed in
AR-driven CRPC bone metastases (Table 2 and Supplemen-
tary Fig. 2) possibly contributed to the low immune-cell
infiltration, and this pathway was therefore selected for
further examination.
3.3. Downregulation of MHC class I expression during PC
progression
To verify the PCA findings, we analyzed the expression
levels of genes involved in MHC class I antigen processing
[(Fig._2)TD$FIG]
Fig. 2 – Immunohistochemical analysis demonstrating (A) significantly higher infiltration of CD68+ immune cells and (B) borderline higher infiltration
of CD3+ immune cells in non–AR-driven (n = 8) compared to AR-driven (n = 26) castration-resistant prostate cancer bone metastases. Sections show
representative (C) CD68 and (D) CD3 staining of a non–AR-driven metastasis sample.
E U R O P E A N U R O L O G Y 7 1 ( 2 0 1 7 ) 7 7 6 – 7 8 7 780