ITGA5 inhibition in pancreatic stellate cells attenuates desmoplasia and potentiates efficacy of chemotherapy in pancreatic cancer

Abstract

Abundant desmoplastic stroma is the hallmark for pancreatic ductal adenocarcinoma (PDAC), which not only aggravates the tumor growth but also prevents tumor penetration of chemotherapy, leading to treatment failure. There is an unmet clinical need to develop therapeutic solutions to the tumor penetration problem. In this study, we investigated the therapeutic potential of integrin α5 (ITGA5) receptor in the PDAC stroma. ITGA5 was overexpressed in the tumor stroma from PDAC patient samples, and overexpression was inversely correlated with overall survival. In vitro, knockdown of ITGA5 inhibited differentiation of human pancreatic stellate cells (hPSCs) and reduced desmoplasia in vivo. Our novel peptidomimetic AV3 against ITGA5 inhibited hPSC activation and enhanced the antitumor effect of gemcitabine in a 3D heterospheroid model. In vivo, AV3 showed a strong reduction of desmoplasia, leading to decompression of blood vasculature, enhanced tumor perfusion, and thereby the efficacy of gemcitabine in co-injection and patient-derived xenograft tumor models.

Fig. 1. ITGA5 expression and prognostic value in PDAC and PSCs.

(A) Immunohistochemical staining for ITGA5 performed on pancreatic tumor and normal pancreas on tissue microarrays (TMAs). Scale bar, 100 μm. (B) Double immunofluorescence staining shows ITGA5 (red) and α-SMA (green) with DAPI (4′,6-diamidino-2-phenylindole; blue nuclei) in the PDAC tissue. Scale bar, 100 μm. (C) Kaplan-Meier overall survival curves for the stromal expression of ITGA5 and α-SMA in patients with PDAC. Log-rank test was performed to calculate significant differences. Survival analyses were performed using the Kaplan-Meier method. (D) Transcriptomic analysis of ITGA5 in publicly available microarray dataset (GSE28735). (E) Immunofluorescence staining showing α-SMA and ITGA5 expression levels in hPSCs with or without TGF-β activation highlighting morphological changes. (F) Western blot analysis and (G) gene expression analysis using quantitative polymerase chain reaction (qPCR) for α-SMA and ITGA5 in hPSCs with or without TGF-β activation. Data represent means ± SEM from at least three independent experiments. *P < 0.05 and ***P < 0.001.

Fig. 2. ITGA5 knockdown attenuates hPSC activation, differentiation, and functions.

Immunofluorescence staining showing ITGA5 (A) and α-SMA (B) expression in hPSCs after ITGA5 knockdown (sh-ITGA5) compared to NC (sh-NC) hPSCs with or without TGF-β activation. (C) Differential gene expression of adhesion molecules and ECM using human ECM RT² Profiler PCR Array in sh-ITGA5 with or without TGF-β activation. Left graph: sh-ITGA5 data are relative to sh-NC (set at 1.0) showing top 20 down-regulated genes after ITGA5 knockdown. Right graph: sh-NC + TGF-β data are relative to sh-NC hPSCs, while sh-ITGA5 + TGF-β data are relative to sh-ITGA5 hPSCs, showing the most induced genes after TGF-β activation, which are not induced after ITGA5 knockdown. (D) Cell adhesion assay shows that ITGA5 knockdown in hPSCs (sh-ITGA5) reduced cell attachment to the coated FN at t = 30 min compared to control hPSCs (sh-NC). Scale bar, 100 μm. (E) Spheroid formation assay shows that sh-ITGA5 hPSCs do not form compact spheroid due to lowered cell-to-cell attachment. (F) BrdU enzyme-linked immunosorbent assay (ELISA) shows that sh-ITGA5 hPSCs had a reduced cell proliferation compared to sh-NC assessed for 3 days. (G) Representative microscopic images of wound closure assay and quantitative analyses at t = 15 hours shows that sh-ITGA5 hPSC had a reduced migration ability. (H) Representative images from 3D collagen gel contractility assay show that TGF-β–induced contractility was inhibited in sh-ITGA5 hPSCs after 96 hours. (I) Western blot analysis (bands and quantitative analysis) shows that sh-ITGA5 hPSCs had a reduced TGF-β–induced pSmad2 and pFAK signaling in comparison to sh-NC hPSCs. The pSmad2/Smad2 ratio was analyzed at t = 30 min, while the pFAK-Y397/FAK ratio was analyzed at t = 48 hours. Densitometry analyses were performed using ImageJ software. Data represent means ± SEM from at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bar, 200 μm. n.d., not determined.

Fig. 3. ITGA5 knockdown abolishes PSC-induced protumorigenic effects.

(A) Schematic representation of the paracrine effect of hPSCs on tumor cells. (B) PANC-1 tumor cell growth after 72 hours of incubation with conditioned medium obtained from sh-NC or sh-ITGA5 hPSCs activated either with or without TGF-β1. Cell growth was determined using alamarBlue assay performed at t = 0 hours and t = 72 hours and presented as % of t = 0 hours. Scale bar, 100 μm. (C) Effect of hPSC on PANC-1 migration. Representative images from transwell insert assay showing the migration of PANC-1 after incubation for 16 hours with conditioned medium (CM) from either sh-NC or sh-ITGA5 hPSCs with or without TGF-β. Data show that TGF-β–activated hPSCs did not induce PANC-1 migration, while there was a reduced migration of PANC-1 with sh-ITGA5 conditioned medium compared to sh-NC conditioned medium. Data represent means ± SEM from at least three independent experiments. *P < 0.001. (D) Tumor growth curves comparing the tumor growth of PANC-1 versus PANC-1 + hPSCs (left graph) and PANC-1 + sh-NC hPSCs versus PANC-1 + sh-ITGA5 hPSCs (right graph). Data represent means + SEM, *P < 0.05, **P < 0.01. (E) Representative microscopic pictures of immunohistochemical staining for α-SMA and collagen I in tumors. Scale bar, 100 μm. (F and G) Quantitation of α-SMA and collagen I staining, respectively. Data represents means + SEM. *P < 0.05, ***P < 0.001.

Fig. 4. A novel peptidomimetic (AV3) against ITGA5.

(A) Chemical structure of AV3 peptidomimetic. (B) Binding of AV3-FAM in control hPSCs, TGF-β–activated hPSCs, and sh-ITGA5 hPSCs, as shown in the representative microscopic fluorescent images and quantitative analysis from flow cytometry. (C and D) Immunocytochemical staining and Western blot analyses show that AV3 inhibits α-SMA and collagen I expression levels in hPSCs, whereas scrambled (s)AV3 shows no inhibitory effects. (E) Bar graph showing the results from the collagen gel assay in which AV3 inhibits TGF-β–induced collagen gel contractility after 72 hours. (F) Western blot analyses showing the expression levels of pFAK, FAK, and β-actin in hPSCs following AV3 treatment of TGF-β–activated hPSCs for 8 hours. (G) Representative images of PANC-1 + hPSC (A) and MIA PaCa-2 + hPSC (H) heterospheroids. The graph shows the spheroid volume and cell viability after the treatment with AV3 and cotreatment with AV3 and gemcitabine compared to either vehicle- or gemcitabine-treated spheroids. Data represent means ± SEM from at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5. AV3 reduces PSC-induced pancreatic tumor growth and potentiates the effect of gemcitabine in co-injection tumor models in mice.

The dataset A–E belongs to PANC-1 + hPSCs, while the dataset F–I belongs to MIA PaCa-2 + hPSCs. (A and F) Tumor growth curve of PANC-1 + hPSC and MIA PaCa + hPSC tumors after the treatment with vehicle, AV3, gemcitabine (Gem), or AV3 + Gem. AV3 was administered at the dose of 20 mg/kg, intraperitoneally, three times per week for only the first week, followed by biweekly, while gemcitabine was administered at the dose of 50 mg/kg, intraperitoneally, biweekly (after the second dotted line). Data are represented as means ± SEM. n = 4 mice per group. *P < 0.05, **P < 0.01, and ***P < 0.001. (B and G) Images of the isolated PANC-1 + hPSC and MIA PaCa + hPSC tumors and (C and H) tumor weights of the isolated PANC-1 + hPSC and MIA PaCa + hPSC tumors at the end of the experiment. (D and I) Optical imaging showing the effect of AV3 on the accumulation of ICG dye in PANC-1 + hPSC and MIA PaCa + hPSC tumors. Mice were injected with ICG dye via tail vein at the dose of 5 mg/kg. After 24 hours of the injection, isolated tumors were imaged using a near-infrared (NIR) animal imager. The degree of ICG fluorescence signal was quantified. n = 4 mice per group. Data represent means ± SEM. *P < 0.05. (E) Microscopic images of immunofluorescence staining of collagen I show that the treatment with AV3 alone or combined with gemcitabine reduced the expression of collagen I. Blood vessel lumens in AV3-treated tumors were decompressed, as shown in (E) (see arrows).

Fig. 6. AV3 potentiates the effect of gemcitabine in the PDX model in mice.

(A) Schematic representation of the generation of the human pancreatic PDX tumor model. (B) Tumor growth curves in the PDX model after the treatment with vehicle, AV3 (20 mg/kg, intraperitoneally, twice a week), gemcitabine, or AV3 + Gem. The doses of AV3 and Gem were 20 and 50 mg/kg, respectively, intraperitoneally, biweekly, starting at day 0. Data represent means ± SEM. n = 5 mice per group. *P < 0.05 and **P < 0.01. (C) Images of the isolated tumors and (D) tumor weights at the end of the experiment. (E) NIR imaging showing the accumulation of ICG dye in tumors treated with either vehicle or AV3. The degree of ICG fluorescence signal was quantified. n = 5 mice per group. Data represent means ± SEM. (F) Microscopic pictures of immunohistochemical staining of collagen I and CD31, and CD31 lumen size quantitation. Data represent means ± SEM. *P < 0.05 and **P < 0.01.