Background Pancreatic ductal adenocarcinoma (PDAC) often fails to respond to immune therapies due to various factors, including the role of Epithelial to Mesenchymal Transition (EMT) plasticity in conferring broad resistance to diverse therapies.1–6 However, the relationship between cancer cell heterogeneity and the tumor immune microenvironment remains unclear. To address this, we utilized single nuclei RNA-seq and spatial transcriptomics to uncover the landscape of these cell-cell interactions in human PDAC,7 which we applied to specimens from two immunotherapy trials.
The first trial is based on TGF-beta being a major driver of EMT.2 8–11 Losartan, an indirect TGF-beta inhibitor,12 13 showed promise with combination chemotherapy FOLFIRINOX in an initial clinical trial.14 A randomized multi-institutional clinical trial of FOLFIRINOX +/- losartan +/- nivolumab (anti-PD1) for PDAC has been completed (NCT03563248).
The second trial focused on exploring the abscopal effect induced by radiation therapy when combined with immune checkpoint inhibition (anti-PD-L1 + anti-CTLA4) in PDAC tumors.15 A pilot trial demonstrated a 29% disease control rate with combined nivolumab + ipilimumab and radiation therapy.16 A follow-up Phase II single-arm study evaluating this combination in metastatic PDAC is completed (NCT04361162).
Methods Using the NanoString GeoMx Digital Spatial Profiler, we selected multiple regions of interest in formalin-fixed paraffin-embedded (FFPE) human PDAC specimens. Immunofluorescent antibody-guided isolation of RNA and protein from cancer cells (pan-cytokeratin), cancer-associated fibroblasts (alpha-SMA), and immune cells (CD45) were performed. Utilizing the whole transcriptome assay (WTA; 18,000+ protein-coding genes) and a new IO Proteome Atlas (IPA; 500+ plex proteins), we ventured to understand the relationship between tumor cells and the surrounding microenvironment.
Results PDAC cells, CAFs, and immune cells were successfully characterized using NanoString GeoMx in clinical trial specimens. Analysis revealed associations between cancer cell plasticity, TGF-beta signaling, and PDAC cell states (Epithelial and Mesenchymal). These differences were observed between Arm 1 (FOLFIRINOX) and Arm 2 (FOLFIRINOX+losartan) in resected neoadjuvant-treated PDAC tumors (figure 1). Immune deconvolution analysis identified variations in immune infiltrates, showing an anti-correlation between macrophages and T-cells (figure 2).
Conclusions Spatial transcriptomics and proteomics reveal insights into the spatial relationship between PDAC tumor cell EMT plasticity, CAFs, and immune infiltrates. This enables the discovery of novel immune response biomarkers and potential therapeutic avenues to target tumor and microenvironment interactions.
Acknowledgements We thank Danielle Bestoso as project manager for the Tumor Cartography Center at the Mass General Cancer Center.
Trial Registration DF/HCC protocol 18–179: Losartan and Nivolumab in Combination With FOLFIRINOX and SBRT in Localized Pancreatic Cancer. NCT03563248
DF/HCC protocol 19–587: Nivolumab + Ipilimumab + Radiation in MSS Pancreatic Cancer NCT04361162
References 1. Porter RL, Magnus NKC, Thapar V, et al. Epithelial to mesenchymal plasticity and differential response to therapies in pancreatic ductal adenocarcinoma [published correction appears in Proc Natl Acad Sci U S A. 2020 Jan 13;:]. Proc Natl Acad Sci U S A. 2019;116(52):26835–26845. doi:10.1073/pnas.1914915116
2. Ligorio M, Sil S, Malagon-Lopez J, et al. Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer. Cell. 2019;178(1):160–175.e27. doi:10.1016/j.cell.2019.05.012
3. Ting DT, Wittner BS, Ligorio M, et al. Single-cell RNA sequencing identifies extracellular matrix gene expression by pancreatic circulating tumor cells. Cell Rep. 2014;8(6):1905–1918. doi:10.1016/j.celrep.2014.08.029
4. Moncada R, Barkley D, Wagner F, et al. Integrating microarray-based spatial transcriptomics and single-cell RNA-seq reveals tissue architecture in pancreatic ductal adenocarcinomas [published correction appears in Nat Biotechnol. 2020 Dec;38(12):1476]. Nat Biotechnol. 2020;38(3):333–342. doi:10.1038/s41587–019-0392–8
5. Peng J, Sun BF, Chen CY, et al. Single-cell RNA-seq highlights intra-tumoral heterogeneity and malignant progression in pancreatic ductal adenocarcinoma [published correction appears in Cell Res. 2019 Aug 13;:]. Cell Res. 2019;29(9):725–738. doi:10.1038/s41422–019-0195-y
6. Single-Cell Transcriptomics of Pancreatic Cancer Precursors Demonstrates Epithelial and Microenvironmental Heterogeneity as an Early Event in Neoplastic Progression. Clin Cancer Res, 2019;25(7):2194–2205.
7. Hwang WL, Jagadeesh KA, Guo JA, et al. Single-nucleus and spatial transcriptome profiling of pancreatic cancer identifies multicellular dynamics associated with neoadjuvant treatment. Nat Genet. 2022;54(8):1178–1191. doi:10.1038/s41588–022-01134–8
8. Zhong Y, Macgregor-Das A, Saunders T, et al. Mutant p53 Together with TGFβ Signaling Influence Organ-Specific Hematogenous Colonization Patterns of Pancreatic Cancer. Clin Cancer Res. 2017;23(6):1607–1620. doi:10.1158/1078–0432.CCR-15–1615
9. Huang W, Navarro-Serer B, Jeong YJ, et al. Pattern of Invasion in Human Pancreatic Cancer Organoids Is Associated with Loss of SMAD4 and Clinical Outcome. Cancer Res. 2020;80(13):2804–2817. doi:10.1158/0008–5472.CAN-19–1523
10. Yu M, Ting DT, Stott SL, et al. RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis [published correction appears in Nature. 2012 Oct 25;490(7421):570]. Nature. 2012;487(7408):510–513. doi:10.1038/nature11217
11. Raghavan S, Winter PS, Navia AW, et al. Microenvironment drives cell state, plasticity, and drug response in pancreatic cancer. Cell. 2021;184(25):6119–6137.e26. doi:10.1016/j.cell.2021.11.017
12. Arnold SA, Rivera LB, Carbon JG, et al. Losartan slows pancreatic tumor progression and extends survival of SPARC-null mice by abrogating aberrant TGFβ activation. PLoS One. 2012;7(2):e31384. doi:10.1371/journal.pone.0031384
13. Chauhan VP, Martin JD, Liu H, et al. Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels. Nat Commun. 2013;4:2516. doi:10.1038/ncomms3516
14. Boucher Y, Posada JM, Subudhi S, et al. Addition of Losartan to FOLFIRINOX and Chemoradiation Reduces Immunosuppression-Associated Genes, Tregs, and FOXP3+ Cancer Cells in Locally Advanced Pancreatic Cancer. Clin Cancer Res. 2023;29(8):1605–1619. doi:10.1158/1078–0432.CCR-22–1630
15. Twyman-Saint Victor C, Rech AJ, Maity A, et al. Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer. Nature. 2015;520(7547):373–377. doi:10.1038/nature14292
16. Parikh, Aparna R, et al. ‘Radiation therapy enhances immunotherapy response in microsatellite stable colorectal and pancreatic adenocarcinoma in a phase II trial.’ Nature cancer 2021;2(11):1124–1135. doi:10.1038/s43018–021-00269–7
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