Article Text

Download PDFPDF

655 Dissecting pancreatic cancer tumor-immune microenvironment crosstalk using spatial transcriptomics
  1. Amaya Pankaj1,
  2. Arnav Mehta2,
  3. Michael J Raabe2,
  4. Julie L Koenig2,3,
  5. Bidish Patel2,
  6. Evan R Lang2,
  7. Joshua Kocher2,
  8. Katherine Xu3,
  9. Linda Nieman2,
  10. Alec Kimmelman4,
  11. David Ryan2,5,
  12. William Hwang2,4,
  13. Martin Aryee6,
  14. Theodore Hong3,
  15. Aparna R Parikh2,3 and
  16. David Ting2
  1. 1Massachusetts General Hospital, Newton, MA, USA
  2. 2Mass General Cancer Center, Harvard Medical School Hospital, Charlestown, MA, USA
  3. 3Massachusetts General Hospital, Boston, MA, USA
  4. 4New York University School of Medicine, New York, NY, USA
  5. 5Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
  6. 6Dana Farber Cancer Institute, Boston, MA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


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

Ethics Approval All studies presented were approved by the Dana-Farber/Harvard Cancer Center IRB protocols 18–179 and 19–587.

Abstract 655 Figure 1

Gene expression heatmap of epithelial (Classical) and mesenchymal (Basal like) genes in tumor cells from patients on the FOLFIRINOX +/- losartan +/- nivolumab trial.

Abstract 655 Figure 2

Immune cell de-convolution from patients on the losartan trial demonstrating relative anti-correlation of T-cells and macrophages across arms

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.