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Single-cell RNA sequencing to characterize the response of pancreatic cancer to anti-PD-1 immunotherapy.

ABSTRACT: Pancreatic cancer (PaC) is resistant to immune checkpoint therapy, but the underlying mechanisms are largely unknown. In this study, we have established four orthotopic PaC murine models with different PaC cell lines by intra-pancreatic inoculation. Therapeutic examinations demonstrate that only tumors induced with Panc02-H7 cells respond to αPD-1 antibody treatment, leading to significantly reduced tumor growth and increased survival in the recipient mice. Transcriptomic profiling at a single-cell resolution characterizes the molecular activity of different cells within tumors. Comparative analysis and validated experiments demonstrate that αPD-1-sensitive and -resistant tumors differently shape the immune landscape in the tumor microenvironment (TME) and markedly altering effector CD8+ T cells and tumor-associated macrophages (TAMs) in their number, frequency, and gene profile. More exhausted effector CD8+ T cells and increased M2-like TAMs with a reduced capacity of antigen presentation are detected in resistant Panc02-formed tumors versus responsive Panc02-H7-formed tumors. Together, our data highlight the correlation of tumor-induced imbalance of macrophages with the fate of tumor-resident effector CD8+ T cells and PaC response to αPD-1 immunotherapy. TAMs as a critical regulator of tumor immunity and immunotherapy contribute to PaC resistance to immune checkpoint blockade.

SUBMITTER: Zhou J

PROVIDER: S-EPMC8591363 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Human pancreatic ductal adenocarcinoma (PDAC) responds poorly to immune checkpoint inhibitor (ICPi). While the mechanism is not completely clear, it has been recognized that tumor microenvironment (TME) plays key roles. We investigated if targeting CD47 with a monoclonal antibody could enhance the response of PDAC to ICPi by altering the TME. METHODS:Using immunohistochemistry, we examined tumor-infiltrating CD68+ pan-macrophages (CD68+ M) and CD163+ M2 macrophages (CD163+ M2) and tumor expression of CD47 and PD-L1 proteins in 106 cases of PDAC. The efficacy of CD47 blockade was examined in xenograft models. CD45+ immune cells from syngeneic tumor models were subjected to single-cell RNA-sequencing (scRNA-seq) by using the 10x Genomics pipeline. RESULTS:We found that CD47 expression correlated with the level of CD68+ M but not CD163+ M2. High levels of tumor-infiltrating CD68+ M, CD163+ M2, and CD47 expression were significantly associated with worse survival. CD47high/CD68+ Mhigh and CD47high/CD163+ M2high correlated significantly with shorter survival, whereas CD47low/CD68+ Mlow and CD47low/CD163+ M2low correlated with longer survival. Intriguingly, CD47 blockade decreased the tumor burden in the Panc02 but not in the MPC-83 syngeneic mouse model. Using scRNA-seq, we showed that anti-CD47 treatment significantly remodeled the intratumoral lymphocyte and macrophage compartments in Panc02 tumor-bearing mice by increasing the pro-inflammatory macrophages that exhibit anti-tumor function, while reducing the anti-inflammatory macrophages. Moreover, CD47 blockade not only increased the number of intratumoral CD8+ T cells, but also remodeled the T cell cluster toward a more activated one. Further, combination therapy targeting both CD47 and PD-L1 resulted in synergistic inhibition of PDAC growth in the MPC-83 but not in Panc02 model. MPC-83 but not Panc02 mice treated with both anti-CD47 and anti-PD-L1 showed increased number of PD-1+CD8+ T cells and enhanced expression of key immune activating genes. CONCLUSION:Our data indicate that CD47 targeting induces compartmental remodeling of tumor-infiltrating immune cells of the TME in PDAC. Different PDAC mouse models exhibited differential response to the anti-CD47 and anti-PD-L1 blockade due to the differential effect of this combination treatment on the infiltrating immune cells and key immune activating genes in the TME established by the different PDAC cell lines.

Project description:PurposeTo define dominant molecular and cellular features associated with PD-1/PD-L1 blockade resistance in metastatic urothelial cancer.Experimental designWe pursued an unbiased approach using bulk RNA sequencing data from two clinical trials to discover (IMvigor 210) and validate (CheckMate 275) pretreatment molecular features associated with resistance to PD-1/PD-L1 blockade in metastatic urothelial cancer. We then generated single-cell RNA sequencing (scRNA-seq) data from muscle-invasive bladder cancer specimens to dissect the cellular composition underlying the identified gene signatures.ResultsWe identified an adaptive immune response gene signature associated with response and a protumorigenic inflammation gene signature associated with resistance to PD-1/PD-L1 blockade. The adaptive immune response:protumorigenic inflammation signature expression ratio, coined the 2IR score, best correlated with clinical outcomes, and was externally validated. Mapping these bulk gene signatures onto scRNA-seq data uncovered their underlying cellular diversity, with prominent expression of the protumorigenic inflammation signature by myeloid phagocytic cells. However, heterogeneity in expression of adaptive immune and protumorigenic inflammation genes was observed among single myeloid phagocytic cells, quantified as the myeloid single cell immune:protumorigenic inflammation ratio (Msc2IR) score. Single myeloid phagocytic cells with low Msc2IR scores demonstrated upregulation of proinflammatory cytokines/chemokines and downregulation of antigen presentation genes, were unrelated to M1 versus M2 polarization, and were enriched in pretreatment blood samples from patients with PD-L1 blockade-resistant metastatic urothelial cancer.ConclusionsThe balance of adaptive immunity and protumorigenic inflammation in individual tumor microenvironments is associated with PD-1/PD-L1 resistance in urothelial cancer with the latter linked to a proinflammatory cellular state of myeloid phagocytic cells detectable in tumor and blood.See related commentary by Drake, p. 4139.

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Single-cell RNA sequencing to characterize the response of pancreatic cancer to anti-PD-1 immunotherapy.

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