Antifibrotic Therapy Disrupts Stromal Barriers and Modulates the Immune Landscape in Pancreatic Ductal Adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDA) remains one of the deadliest forms of cancer, in part, because it is largely refractory to current therapies. The failure of most standard therapies in PDA, as well as promising immune therapies, may be largely ascribed to highly unique and protective stromal microenvironments that present significant biophysical barriers to effective drug delivery, that are immunosuppressive, and that can limit the distribution and function of antitumor immune cells. Here, we utilized stromal reengineering to disrupt these barriers and move the stroma toward normalization using a potent antifibrotic agent, halofuginone. In an autochthonous genetically engineered mouse model of PDA, halofuginone disrupted physical barriers to effective drug distribution by decreasing fibroblast activation and reducing key extracellular matrix elements that drive stromal resistance. Concomitantly, halofuginone treatment altered the immune landscape in PDA, with greater immune infiltrate into regions of low hylauronan, which resulted in increased number and distribution of both classically activated inflammatory macrophages and cytotoxic T cells. In concert with a direct effect on carcinoma cells, this led to widespread intratumoral necrosis and reduced tumor volume. These data point to the multifunctional and critical role of the stroma in tumor protection and survival and demonstrate how compromising tumor integrity to move toward a more normal physiologic state through stroma-targeting therapy will likely be an instrumental component in treating PDA. SIGNIFICANCE: This work demonstrates how focused stromal re-engineering approaches to move toward normalization of the stroma disrupt physical barriers to effective drug delivery and promote antitumor immunity.See related commentary by Huang and Brekken, p. 328.

Figure 1.. Desmoplasia creates stromal barriers and limits drug delivery in PDA.

(A-C) Activated PSCs express α-SMA+, emerge during preinvasive disease, and remain prevalent throughout PDA. (D-F) Masson’s trichrome staining shows collagen (blue) deposition by myofibroblasts during disease. (G-I) Fibrillar collagen determined from second harmonic generation imaging. (J-L) Intense hyaluronan expression during disease. (M-O) CD31 immunohistochemistry shows transition from open vessels in normal pancreata (arrowheads) to increasingly showing the phenotype of vascular collapse (arrows). (P-R) Decreased doxorubicin distribution in disease (after i.v. injection 15–20 minutes before euthanasia). S) PDA is hypovascular (n>6 regions/condition). (T) Extremely elevated interstitial fluid pressure (IFP) in PDA (n=4/group). (U) Key ECM and TGF-β pathway transcripts (n=178 TCGA patients) are upregulated in human PDA. (V) Strong correlation between α-SMA (ACTA2) and collagen-I (COL1A1) expression in human PDA. (W) Collagen expression relates to survival in human patient (TCGA PDA dataset). (X-Z) Human TCGA data shows Strong correlation between genetic collagen-I expression and key profibrotic TGF-β signaling cascade components. (Scale bar=25μm, p****<0.0001, Data are mean±SEM)

Figure 2.. Halofuginone inhibits fibrotic activity in vitro and in vivo.

(A-C) Immunofluorescent staining of DAPI (blue) and α-SMA (green) in murine (B) and human (C) PSCs shows dose-dependent inhibition of α-SMA (scale bar=10μm; n=3/condition with 5 ROIs analyzed/sample). (D) Expression (via qPCR) of the fibrosis-related genes Col1a1 (collagen-I) and Has2 (hyaluronan synthase-2) are significantly inhibited with increasing HF concentration. (E) Dose-dependent proliferation inhibition (MTS proliferation assay) in murine (mPSC) and human PSCs (hPSC) following 48 hours of HF treatment. (F,G,N) In vitro observations are maintained in vivo, where α-SMA immunohistochemical reveals a decrease in PSC activation (α-SMA+) with HF. (H-Q) Consistent with these results, there was a Significant decrease in hyaluronan (H,I,O) and collagen (via Trichrome staining (J,K,P) and SHG for fibrillar collagen (L,M,Q)). (n=6 control; n=5 HF-treated mice;10 ROIs analyzed/tumor) (Scale bar=50 μm, p*<0.05, p**<0.01, p***<0.001, p****<0.0001, Data are mean±SEM)

Figure 3.. Antifibrotic therapy facilitates drug delivery in autochthonous PDA.

(A) Decreased IFP following antifibrotic therapy (n=4 WT; n=4 Control KPC, n=3 HF-treated KPC). (B,C) CD31 immunohistochemistry from control and HF-treated KPC tumors. (D,E) Vessel perfusion with FITC-conjugated dextrans (100μL of 10mg/mL solution injected intracardiac 5 minutes prior to euthanasia). (F) Improved vessel perfusion following HF treatment (perfused vessels/total vessel; n=3 mice/group; ≥5 ROI assessed/tumor). (G-I) Clear recovery of doxorubicin distribution (following i.v. injection 15–20min before euthanasia) in the HF-treated tumors (I), in contrast to little to no perfusion in control tumors (H). (J-L) Significantly reduced fibrillar collagen density and organization (via SHG) (M-O) Merging doxorubicin and SHG data illustrates improved drug distribution correlating with lower collagen density. (P,Q) Quantification of doxorubicin distribution (P) and collagen (Q)(n=3 mice/group; 5 ROI assessed/tumor). Scale bar=50 μm, p*<0.05, p**<0.01, p***<0.001, p****<0.0001, Data are mean±SEM.

Figure 4.. Halofuginone inhibits proliferation of carcinoma cells without enhancing chemotherapy.

(A,B) Dose-response curve of HF-treated (A) or gemcitabine-treated (B) primary murine carcinoma cells (mPDA). (C) Proliferation assay timeline to test ability of HF to sensitize cells to gemcitabine. (D) Results of Sensitization assay showing that the relative response to gemcitabine remains largely static (~66% decrease in proliferation), regardless of HF pre-treatment concentration, indicating that HF does not increase sensitivity of carcinoma cells to gemcitabine (n=3 replicates/condition).

Figure 5.. Antifibrotic therapy modulates the immune landscape.

(A-C) Histopathology analysis of KPC tumor H&E sections suggests a dramatic increase in immune infiltrate 24hr after HF treatment (blowup panels: arrows highlight immune cells; arrowheads highlight larger carcinoma cell nuclei associated). (D) Quantification of immune infiltrate in H&E sections confirming a significant influx of immune cells 24hrs after HF treatment (determined using an algorithm to detect histologic immune infiltrate by filtering nuclei on the basis of size, shape, and density; n=3 mice/group; ≥3 ROI/tumor). (E) Significant Cd11b+ cell increases after 24hrs and 10d revealing that the majority of immune infiltrate is myeloid cells. (F) Quantification of Ly6G+ cells, a validated marker for immunosuppressive CD11b+ MDSCs in PDA, showed no significant difference between treatment groups. (G) Quantification of iNos+ cells, a functional marker for classically activated macrophages with inflammatory functions, shows significant increases after HF treatment. (H, I) No significant difference in (H) CD3⁺;CD4+ T helper cells or (I) CD4+;FoxP3+ Treg cell populations. (J) Quantification of CD3+;CD8+ dual IF showing a significant increase in cytotoxic CD8+ T cells in tumors treated for 24 hours. (K) Percent FOVs with at least one CD8⁺ T cell, showing a significant increase in the distribution of CD8+ T cells throughout PDA tumors. For E-K n≥5 control, n=3 24hr, n=5 10d mice with ≥4 ROI/tumor. Scale bar = 50 μm, p*<0.05, p****<0.0001, Data are plotted as mean±SEM.

Figure 6.. Immune infiltrate in PDA correlates with regions of decreased Hyaluronan

(A-C) H&E staining of untreated (A) and HF-treated (B,C) KPC tumors and adjacent sections stained for hyaluronan (D-F), showing regions of histologically-identifiable immune infiltrate have lower hyaluronan. In addition, following HF treatment hyaluronan-low regions are larger and more frequent (Asterisk: example of lower HA regions that are immune cell rich). (G-I) SHG analysis shows a collagen matrix loosening (i.e. increased fiber waviness, or crimp, associated with low stress conditions) 24hr after HF treatment and decreased collagen at 10d. (J) Collagen fraction analysis from SHG imaging showing a slight increase 24h after HF treatment, consistent with increased waviness, and a significant decrease after 10 days. (K) Significant decreases in hyaluronan following HF treatment (n≥3/condition with ≥5 ROI assessed per tumor). (L) Significant, moderate, correlation (p<0.006; r=0.−46) between hyaluronan (HA) area fraction and immune cell numbers showing increasing immune cell number with lower HA. (M) Comparison of immune cells in low and high HA regions (bottom and top 50% of signal for each group, respectively) showing greater immune cell numbers in low vs. high regions, and significantly increased immune infiltrate cells after HF treatment, consistent with increased low HA regions following HF treatment. (p*<0.05, p**<0.01, p***<0.001. Scale bar A-F = 100 μm; Scale bar G-I = 50 μm; data are plotted as mean±SEM)

Figure 7.. Halofuginone induces widespread necrosis and inhibits tumor volume growth.

(A-D) 3D reconstructed tumor volumes in control versus HF-treated KPC mice. (E) Quantification of the relative change in 3D tumor volume following one week of treatment (n=5 control; n=7 HF). (F,G) Intratumoral necrosis in a control versus HF-treated KPC tumor after 10 days (green=PDA region, red=necrotic region. (H) Quantification of percent necrosis observed within the defined PDA tumor region at 10-day study endpoint (n=9 Control; n=6 HF). Scale bar = 2 mm, p*<0.05, data are mean±SEM.