(M1330-04-19) Stromal Fibrin Inhibition Shapes Immune Infiltration Landscape of Pancreatic Ductal Adenocarcinoma

Purpose: The immune-depleted microenvironment of PDAC, characterized by dense fibrotic stroma and differential infiltration of myeloid-derived immune cells, contributes to its suppressive tumor microenvironment (TME)¹. PDACs often contain small fibrin clots (crosslinked fibrin) into their vasculature and stroma. Fibrinogen, a key coagulation factor that polymerizes to form crosslinked fibrin, is found in excessive amounts in the stroma of PDAC from early to late stages, indicating an early activation of the coagulation cascade during progression. High levels of fibrin clots in tumor stroma correlate with poor survival in solid tumors like lung adenocarcinomas^2,3. We hypothesize that crosslinked fibrin protects PDAC against natural killer cells by creating a physical barrier, whereas inhibition of fibrin-crosslinking primes the immunosuppressive PDAC TME.

Methods: Analyzing transcriptome of PDAC patients, we observed the clinical correlation between fibrin-clot relevant genes (FGA, FGB, and FGG) with overall survival. To show clinical correlation, we performed multiplex analysis of tumor tissues from PDAC patients (N =14), characterize them with higher and lower fibrin expression and correlated the abundance of tumor fibrin-clot with plethora of immune cells in the tumor stroma. Proximity was assessed by quantifying the spatial distribution abundance of tumor associated macrophages (TAM; M1 and M2) and CD8 T-cells within a 30 µm radius of PDAC cells. To check the immunomodulator function of fibrin-laden stroma, we prepared Peak 2 Fibrin (P2F) gels and cultured human U937 monocytes in P2F gels with or without conditioned media of pancreatic cancer cells. We then used “photolithography” to engineer a patterned hydrogel with alternating fibrin-positive (P2F+) and collagen-positive (Col+) stripes, closely mimicking the natural variations of PDAC TME. Migration in our “step” gels consisting of alternating P2F+ and Col+ bars, after coating with 10 μg/mL of fibronectin to enable cell adherence, was determined by quantifying the number of cells accumulated on P2F+ versus Col+ stripes at 24 hours after plating, referred as “Migrating index (MI)”. Using a microfluidic PDAC-on-a-chip model, we determined the role of fibrin in TME immune landscape focusing on macrophage infiltration and differentiation. In preclinical studies, using a fibrin-crosslinking inhibitor, A108, we tested the how stromal fibrin inhibition alters the immune cells infiltration in the murine model of PDAC.

Results: Clinical survival data from KM plotter showed that higher expression FGA (p=0.08), FGB (p=0.001), and FGG (p=0.0000349) correlated with poor patient survival of PDAC. We performed multiplex staining of T-cells colocalization: CD4, CD8, FOXP3, PD-L1, PD1 and macrophage colocalization: CD68, CD163, MHCII, CD11c, CD56 along with Cytokeratin and nuclei in the same tissues. We observed that Fibrin^low PDAC tissues had higher abundance of CD8⁺ Tcells than Fibrin^high PDAC tissues. Higher number of M2 (CD163⁺/MCHII^-) than M1 (CD68⁺/MHCII⁺/CD163^-) macrophages in Fibrin^high PDAC tissues were observed. These data suggest a role of fibrin deposition in PDAC immunosuppressive TME. Phenotype mapping and spatial proximity analysis demonstrated that in Fibrin^high areas, M2 TAMs were found in considerably larger quantities around the PDAC cells than in Fibrin^low areas. In Fibrin^low areas, however, the closeness of CD8⁺ Tcells-to-tumor cells was higher (Fig 1). These results suggest that fibrin-rich areas promote protumorigenic TAM infiltration while suppressing infiltration of anti-tumorigenic CD8⁺ Tcells. We also observed that CD163+ M2 TAMs reside in fibrin-laden stroma than areas of collagen in human PDAC tissues, suggesting fibrin-laden stroma initiates monocyte/macrophage migration. We next assessed the MI of multiple human pancreatic cancer, fibroblasts, monocyte/macrophage cell types in an unbiased manner in our patterned step gels with alternating P2F+ and Col+ stripes. Assessment of single cell migration revealed that monocytes/M2 TAMs, but not cancer cells or CAFs, migrate when the step gels were made of fibrin (MI > 4) (Fig 2). In PDAC-on-a-chip model, monocyte-differentiated macrophages, when cultured in collagen gels into one compartment along with PDAC in P2F gels into the side compartment, induced M2 TAM phenotype (CD68⁺/CD163⁺), infiltrated into the PDAC/P2F compartment, and increased the survival of PDAC cells. However, the same phenomenon was not observed when PDAC was grown in the absence of P2F gels. The number of M2 TAM traveling more than 25 µm was high in conditions with PDAC/P2F which also correlate with spatial proximity analysis in clinical tissues. To assess the functional effects of crosslinked fibrin inhibition, we used A108, a peptidomimetic inhibitor, to block insoluble fibrin formation in tumors, A108 increased cytotoxic CD8⁺ T-cells and M1 TAM infiltration, but it decreased the immunosuppressive Treg and M2 TAM in the tumors, indicating that intratumoral clots may create a microenvironment conducive to tumor immune suppression. Subsequent spatial pattern analysis using “R” reveal that the distant between CD8⁺ T-cells and nearest tumor cell was lower in A108-treated tumors than control, suggesting that intratumoral clots restrict CD8⁺ T-cells infiltration (Fig 3).

Conclusion: Our data strongly suggest that fibrin(ogen) actively modulates the immune microenvironment of PDAC, significantly influencing the immune cells infiltration landscape–particularly Tcells and TAMs–contributing to the tumor's immunosuppressive conditions.

References: 1. Ho, W. J., Jaffee, E. M. & Zheng, L. The tumour microenvironment in pancreatic cancer — clinical challenges and opportunities. Nat. Rev. Clin. Oncol. 17, 527–540 (2020).
2. Wang, M. et al. Fibrinogen Alpha Chain Knockout Promotes Tumor Growth and Metastasis through Integrin–AKT Signaling Pathway in Lung Cancer. Mol. Cancer Res. 18, 943–954 (2020).
3. Porrello, A. et al. Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking. Nat. Commun. 9, 1988 (2018).


Fig 1: Correlation of the abundance of tumor fibrin-clot with plethora of immune cells in the tumor stroma


Fig 2: Fibrin gradients specifically induce monocyte/macrophage migration.


Fig 3: Stromal fibrin inhibition alters the immune cells infiltration.