Targeting IRE1a reprograms the tumor microenvironment and enhances anti-tumor immunity in prostate cancer [scRNA-seq2]

Unfolded protein response (UPR) is a central intracellular stress response pathway that is hijacked by tumor cells for their survival. However, how activation of UPR in cancer cells may shape the tumor microenvironment (TME) remains largely unexplored. Here, we investigated the potential role of IRE1a signaling on modulation of TME dynamics in prostate cancer (PCa). We found that IRE1a is increased in PCa patient tumors and genetic inhibition of IRE1a in syngeneic mouse PCa models, as well as in an orthotopic model, dramatically reduced tumor growth. Multiomics analysis suggested that IRE1a ablation in cancer cells significantly potentiated interferon (IFN) response and activation of immune system related pathways in the TME. Single-cell RNA-sequencing (scRNA-seq) revealed that the abundance of immunosuppressive cells, such as tumor-associated macrophages (TAMs), were markedly reduced in the IRE1a deficient tumors. Geneset Enrichment Analysis demonstrated that IFN response was significantly enriched in TAMs, cancer cells, and dendritic cells. Notably, the small molecule IRE1a inhibitor MKC8866 (ORIN1001), currently in clinical trials, reprogrammed the TME and enhanced the response to anti-PD-1 blockade therapy in syngeneic PCa mouse models. Furthermore, a novel scRNA-seq-derived TAM gene signature is strongly associated with poor PCa survival, which is reduced by the MKC8866 + anti-PD-1 combination therapy. Our findings indicate that activation of IRE1a signaling not only promotes cancer cell growth and survival, but it also interferes with anti-tumor immunity in the TME. Thus, IRE1a targeting could present a novel approach for improving anti-PD-1 immunotherapy in PCa. Overall design: scRNA-sequencing (scRNA-seq) was performed on PTEN KO Myc-CaP tumors from animals treated with indicated treatments. Freshly dissected tumor samples were washed with PBS and transferred into GEXSCOPE Tissue Preservation Solution (Singleron), and stored on ice. Six tumor samples from each group were pooled for scRNA-seq. The tissue samples were then washed with Hanks balanced salt solution three times and minced into small pieces which were digested with Tissue Dissociation Solution (Singleron) at 37?°C for 15?min with agitation. After digestion, cells were passed through a 40 µm strainer to remove cell debris and centrifuged at 300?×?g for 5?min. The supernatant was removed and cell pellet was resuspended in 1 ml PBS, followed by red blood cell lysis using RBC lysis buffer. Next, cells were centrifuged at 500?×?g for 5 min and resuspended in PBS. The single cell suspension was adjusted to a concentration of 1?×?105 / ml for library preparation. This was loaded onto microfluidics chips (GEXSCOPE Single Cell RNA-seq Kit, Singleron Biotechnologies) and GEXSCOPE 3´SD scRNAseq libraries were prepared according to manufacturer instructions (Singleron Biotechnologies). Individual libraries were diluted and pooled for next generation sequencing on Illumina NovaSeq with 150 bp paired-end reads.