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Cancer Technologies

Cancer Technologies - Poster Session B

Poster H1 - Combining iterative indirect immunofluorescence imaging with artificial intelligence profiling software to characterize the Pericyte-to-Fibroblast Transition in Pancreatic Ductal Adenocarcinoma

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  

Introduction: Pericytes are stromal cells that perform a variety of roles regulating angiogenesis, vascular stability, and capillary permeability. In recent years, studies have indicated that pericytes can undergo pericyte-to-fibroblast transition (PFT) giving rise to fibroblasts that exhibit genetic markers of pericytes [1]. Our recent study monitoring lineage-traced pericytes in a murine model of lung fibrosis revealed that PFT occurs in response to inflammation-induced injury [2]. Due to these observations and the high heterogeneity of pericytes based on tissue origin [3], we asked if pericytes in pancreatic tissue contribute to inflammatory cancer-associated fibroblasts (iCAFs) in pancreatic ductal adenocarcinoma (PDAC) by undergoing PFT. We chose to study PFT in PDAC because fibroblasts comprise up to 80% of PDAC tumors; therefore, understanding fibroblast origins is important to developing strategies for inhibiting tumor growth [4]. We hypothesized that pericytes can undergo PFT in PDAC and give rise to inflammatory cancer-associated fibroblasts (iCAFs) and that the number of pericyte-derived iCAFs will increase with PDAC progression. We tested this hypothesis by implanting murine PDAC cells in an inducible transgenic mouse model in which pericytes and their progeny are lineage-traced by the expression of TdTomato. Indirect immunofluorescence imaging (4i) was used to identify pericytes and pericyte-lineage cells expressing the iCAF marker, podoplanin, at different time points throughout tumor progression [5, 6].

Materials and

Methods:  

Methods: Genetically engineered Myh11-CreERT2 ROSA STOP-floxed TdTomato tamoxifen-inducible pericyte lineage tracing mice, whose pericytes and their progeny are permanently labeled with the fluorescent TdTomato reporter following a 2-week tamoxifen diet, were orthotopically implanted with cells from KPC mice, an established and clinically relevant model of PDAC. Tumors were harvested on days 3, 18, or 24. 4i was used with DAPI, TdTomato/RFP, and podoplanin to immunolabel cell nuclei, lineage-traced pericytes, and iCAFs, respectively. Confocal images were collected and analyzed with ImageJ (Figure 1, B). iCAFs derived from pericytes that underwent PFT were identified by double-positive expression of TdTomato and podoplanin.

Results, Conclusions, and Discussions: Results and

Discussion: The ability to immunostain and image the same sample with 4i allowed us to compare a sample against itself between staining iterations and lowered the significance of contaminating background features present between each iteration (Figure 1, A). Further work will include the implementation of an agent-based modeling system to simulate the proliferation of pericytes and their contribution to iCAF populations. As a first step to understanding how PFT contributes to iCAF populations, we quantified the number of pericytes in the healthy pancreas, primary KPC tumors, liver metastases, and healthy livers (Figure 1, C). We found that there were elevated numbers of pericytes in the KPC tumors and liver metastases. We observed the presence of TdTomato-positive cells expressing podoplanin, the iCAF marker. Future work will quantify their numbers at different time points during tumor progression and metastases. As one of the first studies to look at lineage-traced pericytes in the pancreas as a source of cancer-associated fibroblasts in PDAC, we have identified a potential cellular target for future chemotherapies.

Acknowledgements (Optional):  References

1. Bartoschek, M., Oskolkov, N., Bocci, M. et al. Spatially and functionally distinct subclasses of breast cancer-associated fibroblasts revealed by single cell RNA sequencing. Nat Commun 9, 5150 (2018). https://doi.org/10.1038/s41467-018-07582-3

2. R.T. Hannan, A.E. Miller, R.C. Hung, C. Sano, S.M. Peirce, T.H. Barker Extracellular matrix remodeling associated with bleomycin-induced lung injury supports pericyte-to-myofibroblast transition Matrix Biol. Plus, 10 (2020), Article 100056, https://doi.org/10.1016/j.devcel.2011.07.001

3. Lavie, D., Ben-Shmuel, A., Erez, N. et al. Cancer-associated fibroblasts in the single-cell era. Nat Cancer 3, 793–807 (2022). https://doi-org.calpoly.idm.oclc.org/10.1038/s43018-022-00411-z

4. Fiori, M.E., Di Franco, S., Villanova, L. et al. Cancer-associated fibroblasts as abettors of tumor progression at the crossroads of EMT and therapy resistance. Mol Cancer 18, 70 (2019). https://doi.org/10.1186/s12943-019-0994-2

4i process 

5. Kramer BA, Del Castillo JS, Pelkmans L, Gut G. Iterative Indirect Immunofluorescence Imaging (4i) on Adherent Cells and Tissue Sections. Bio Protoc. 2023 Jul 5;13(13): e4712. doi: 10.21769/BioProtoc.4712. PMID: 37449033; PMCID: PMC10336569.

6. Schindelin, J., Arganda-Carreras, I., Frise, E. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676–682 (2012). https://doi-org.calpoly.idm.oclc.org/10.1038/nmeth.2019





Acknowledgements

Research reported in this study was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number 1T34GM149492-01.