Fig 2: eIF1A knockdown and HHT suppresses PCa growth by alleviating hypoxia and enhancing the immune response(A) Quantitative reverse-transcription PCR analysis of the relative expression level of HIF1A in the si-NC, si-eIF1A, sh-NC, sh-EIF1A, PBS, and HHT groups. Data were presented as the mean ± SD (3 biological replicates for each group and three technical replicates for each biological replicate; two-sided t tests).(B) Western blot analysis to compare the protein levels of HIF-1α and eIF1A among the si-NC, si-eIF1A, sh-NC, sh-EIF1A, PBS, and HHT groups (3 biological replicates for each group).(C) Western blot analysis to compare the protein levels of HIF-1α and eIF1A upon MG132 treatment and eIF1A knockdown (3 biological replicates for each group).(D) Changes in polysome profiles and polysome-to-monosome (P/M) ratios upon EIF1A knockdown. Data were presented as the mean ± SD (3 biological replicates for each group; two-sided t tests).(E) qPCR analysis comparing the HIF1A mRNA level in polysome and non-polysome fractions upon EIF1A knockdown. Data were presented as the mean ± SD (3 biological replicates for each group and three technical replicates for each biological replicate; two-sided t tests).(F) Quadrant plot illustrating the levels of RPFs and RNA expression levels derived from Ribo-seq, where the larger pink dots represent genes with significantly differential RPF levels (2 biological replicates for each group).(G) GSEA of the differentially activated pathways between the EIF1A knockdown and negative control groups based on translational efficiency derived from Ribo-seq.(H) Comparisons of mouse tumor growth curves and tumor volumes between the HHT + ADT and vehicle groups. Data are presented as mean ± SEM (5 mice for each group). Statistical significance was determined by assessing the group × time interaction via a linear mixed-effects model (restricted maximum likelihood).(I) GSEA of the differentially activated pathways between the HHT + ADT and vehicle groups based on RNA-seq (3 mice for each group).(J) Immune cell inference based on RNA-seq data comparing the tumor purity, immune score, and T cell and macrophage ratio between the HHT + ADT and vehicle groups based on RNA-seq (3 mice for each group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.(K and L) Representative mIF images showing the colocalization of eIF1A+ LE (CK8, eIF1A) and hypoxia markers (HIF-1α, CA9), T cells (CD3), and macrophages (CD68) in subcutaneous Myc-CaP tumors in the HHT+ADT and vehicle groups (4 mice for each group; two-sided t test; also see Figures S9F and G). Scale bars, 200 μm.

Fig 3: eIF1A+ LE-centered “cold” immune microenvironment(A) Heatmap showing the components of 20 identified CNs.(B) Dot plot showing the associations between the proportion of CNs and clinical features. Size of the dots corresponds to significant levels, while the color indicates in which group the CN proportions were elevated (corresponding to the group colors shown on the right).(C) Representative IMC images showing the distributions of CN1, CN7, CN10, and CN15 in different types of PCa. Scale bars, 200 μm.(D) Dot plot showing the associations between the distance from eIF1A+ LE to immune cells and clinical features. Size of the dots corresponds to significant levels, while the color indicates in which group the distances were elevated (corresponding to the group colors shown on the right).(E) Representative IMC images showing the distance from eIF1A+ LE to PD-1- T cells and CD163− macrophages in different types of PCa. Scale bar, 200 μm.(F) Representative mIF images showing the colocalization of eIF1A+ LE (CK8, eIF1A), T cells (CD3, CD8), and macrophages (CD68) in different types of PCa (n = 30, 17, 17, and 13 for LgPAC, HgPAC, IDC, and DAC, respectively). Scale bars, 200 μm.(G) The differences in the ratio of CD8+ T cells and macrophages in a 350 × 200-μm window among different types of PCa (two-sided Wilcoxon tests).(H) Representative IMC images showing the locations of HIF-1α+ patches, as well as the eIF1A+ LE, PD-1− T cells and CD163− macrophages in HIF-1α+ patches among different types of PCa. Scale bar, 200 μm. eIF1A+ LE, PD-1− T cells, CD163− macrophages, and other cells within HIF-1α+ patches were labeled blue, red, orange, and cyan, respectively. The cells outside HIF-1α+ patches were labeled gray.(I) Boxplots comparing the proportions of eIF1A+ LE, CD163− macrophages, and PD-1- T cells inside and outside HIF-1α+ patches. The data points represent individual ROIs, the boxes represent the IQR with the median line inside, and the whiskers show the values within 1.5×IQR (two-sided paired Wilcoxon tests).