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. 2022 Jan;71(1):185-193.
doi: 10.1136/gutjnl-2020-322493. Epub 2021 Jan 11.

Placental growth factor promotes tumour desmoplasia and treatment resistance in intrahepatic cholangiocarcinoma

Shuichi Aoki #  1   2 , Koetsu Inoue #  1   2 , Sebastian Klein  1   3 , Stefan Halvorsen  4 , Jiang Chen  1   5 , Aya Matsui  1 , Mohammad R Nikmaneshi  1 , Shuji Kitahara  1   6 , Tai Hato  1   7 , Xianfeng Chen  8 , Kazumichi Kawakubo  1 , Hadi T Nia  1   9 , Ivy Chen  1   10 , Daniel H Schanne  1 , Emilie Mamessier  1   11 , Kohei Shigeta  1   12 , Hiroto Kikuchi  1   12 , Rakesh R Ramjiawan  1 , Tyge Ce Schmidt  1 , Masaaki Iwasaki  1 , Thomas Yau  13 , Theodore S Hong  14 , Alexander Quaas  3 , Patrick S Plum  15 , Simona Dima  16 , Irinel Popescu  16 , Nabeel Bardeesy  17 , Lance L Munn  1 , Mitesh J Borad  8 , Slim Sassi  4   18 , Rakesh K Jain  1 , Andrew X Zhu  17   19 , Dan G Duda  20
Affiliations

Placental growth factor promotes tumour desmoplasia and treatment resistance in intrahepatic cholangiocarcinoma

Shuichi Aoki et al. Gut. 2022 Jan.

Abstract

Objective: Intrahepatic cholangiocarcinoma (ICC)-a rare liver malignancy with limited therapeutic options-is characterised by aggressive progression, desmoplasia and vascular abnormalities. The aim of this study was to determine the role of placental growth factor (PlGF) in ICC progression.

Design: We evaluated the expression of PlGF in specimens from ICC patients and assessed the therapeutic effect of genetic or pharmacologic inhibition of PlGF in orthotopically grafted ICC mouse models. We evaluated the impact of PlGF stimulation or blockade in ICC cells and cancer-associated fibroblasts (CAFs) using in vitro 3-D coculture systems.

Results: PlGF levels were elevated in human ICC stromal cells and circulating blood plasma and were associated with disease progression. Single-cell RNA sequencing showed that the major impact of PlGF blockade in mice was enrichment of quiescent CAFs, characterised by high gene transcription levels related to the Akt pathway, glycolysis and hypoxia signalling. PlGF blockade suppressed Akt phosphorylation and myofibroblast activation in ICC-derived CAFs. PlGF blockade also reduced desmoplasia and tissue stiffness, which resulted in reopening of collapsed tumour vessels and improved blood perfusion, while reducing ICC cell invasion. Moreover, PlGF blockade enhanced the efficacy of standard chemotherapy in mice-bearing ICC. Conclusion PlGF blockade leads to a reduction in intratumorous hypoxia and metastatic dissemination, enhanced chemotherapy sensitivity and increased survival in mice-bearing aggressive ICC.

Keywords: cholangiocarcinoma; hepatic fibrosis.

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Conflict of interest statement

Competing interests: IC is an employee of STIMIT. TY has served in a consulting or advisory role for Bristol Myers Squibb. RKJ received honorarium from Amgen and consultant fees from Chugai, Ophthotech, Merck, SPARC, SynDevRx. RKJ owns equity in Accurius, Enlight, SPARC, and SynDevRx, and serves on the Boards of Trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund and Tekla World Healthcare Fund. AXZ is a consultant/advisory board member for Bayer. DGD received consultant fees from Bayer, Simcere, Surface Oncology and BMS and research grants from Bayer, Exelixis and BMS. No reagents or support from these companies was used for this study.

Figures

Figure 1
Figure 1
Expression of PlGF in human intrahepatic cholangiocarcinoma (ICC). (A) Representative in situ hybridisation (PlGF-ISH, red dots) staining of PlGF in ICC human tissue. (B) Same image of (A) showing the overlay of classifying cells according to cell type (red, PlGF-positive CAF; yellow, PlGF-negative CAF; grey, PlGF-negative ICC cell). (C) Representative serial section showing PDGFRβ staining (arrow highlights positive cell) to identify fibroblasts. (D) Representative serial section showing AE/AE3 staining (arrow highlights positive cell) to identify cancer cells. (E) Representative serial section showing CD31 staining (DAB, arrow highlights positive cell) of endothelial cells (scale bar 250 µm). (F) Differential levels of the Nrp1 and PGF gene expression calculated by subtracting logRPKM for human (ICC cells) from mouse (stromal cells) in a collection of 22 patient-derived xenograft (PDX) ICC tissues (online supplemental table S3; note the significantly higher expression levels in murine vs human PGF, *p<0.05). CAFs, cancer-associated fibroblasts; PlGF, placental growth factor;
Figure 2
Figure 2
Single cell RNA sequencing analysis of gene expression changes after PlGF blockade. (A) tSNE plot (each dot represents a cell) demonstrating different populations of CAFs and ICC cells based on transcriptional profiles in whole tumour lysate from orthotopic 425 ICC model cultured in vitro with anti-PlGF antibody. The CAF_2 (arrow) cluster was strongly enriched after PlGF blockade. (B) Expression of the PGF gene (encoding for PlGF) across CAF and ICC cell clusters. (C) Proportion plot demonstrating representation of treated and control cells in each cluster. (C–E) The cluster of cells affected by PlGF blockade showed a gene expression pattern associated with increased expression in genes related to hypoxia (hyp) (C) glycolysis (glyc) (D) and Akt pathways (path) (E). *P<0.05; **p<0.001; ***p<0.0001. CAF, cancer-associated fibroblast; ICC, intrahepatic cholangiocarcinoma; PIGF, placental growth factor; tSNE, t-distributed stochastic neighbor embedding.
Figure 3
Figure 3
PlGF promotes a myofibroblast-like phenotype in CAFs via increased NF-kB/Akt activation. (A, B) RNAseq analysis of two CAF clones (A) comparing high α-SMA-expressing myCAF versus low α-SMA-expressing qCAF versus 425 cells (B) (see online supplemental dataset 1). (C) GSEA shows enrichment in NF-kB pathway-related genes in qCAF cells versus control (425 ICC cells). (D) High protein expression of Nrp1 but not Nrp2 or VEGFR1 in CAFs. (E) Recombinant PlGF further activates NF-kB/Akt axis and increases α-SMA expression in qCAFs, while PlGF blockade using 5D11D4 (5D11) treatment downregulates NF-kB/Akt pathway and α-SMA expression. Moreover, inhibition of PI3K using the compoundBKM120 and NF-kB with the compound QNZ also inhibited activation of NF-kB and α-SMA expression. (F) Both PlGF blockade and PI3K inhibition reduced the expression of profibrotic genes in qCAFs (analysed in triplicate). CAF, cancer-associated fibroblast; PIGF, placental growth factor.
Figure 4
Figure 4
Impact of PlGF inhibition on ICC cell invasion. (A–D) Representative images of in vitro 3-D coculture of 425 ICC cells with GFP-expressing qCAFs versus ICC cells alone (A); CAFs increased the spheroid formation compared with 425 ICC cells alone, and this effect was suppressed by PlGF blockade with 5D11D4 (5D11) antibody (B); Scale bar, 160 µm. In addition, 3-D co-culture of ICC cells with CAFs (C) further increased ICC cell invasion, and PlGF blockade with 5D11 antibody inhibited this effect (D). (See online supplemental movie S1). (E, F) Analysis of ICC cells alone in 3-D (spheroid) invasion assays (E) showed that PlGF knockdown or anti-PlGF treatment using 5D11D4 (5D11) inhibited 425 ICC cell invasion in Matrigel (F); this effect was rescued by addition of recombinant (r)PlGF exposure in PlGF knockdown cells (see online supplemental movie S2). All experiments were performed in triplicate; p values from Tukey’s test. CAF, cancer-associated fibroblast; ICC, intrahepatic cholangiocarcinoma; PlGF, placental growth factor.
Figure 5
Figure 5
Impact of genetic inhibition of PlGF in ICC cells on ICC growth and/or pharmacologic blockade of PlGF on established tumour progression. (A, B) PlGF knockdown in ICC cells delayed tumour onset, while complete pharmacological blockade significantly delayed established ICC growth in the orthotopic 425 model (individual growth curves in (A) and average tumour size with SD in surviving mice in (B)). (C, D) Representative IHC for Ki-67 as a marker of cell proliferation in tumour sections from the four treatment groups (C); PlGF blockade—but not PlGF knockdown in ICC cells—significantly reduced in vivo ICC cell proliferation in size-matched orthotopic 425 tumours (D). (E) PlGF blockade significantly reduced the occurrence of bloody ascites. (F, G) Combined genetic and pharmacologic inhibition of PlGF reduced metastasis to the lymph nodes (F) and lungs (G). P values from Mann-Whitney U test (A, B), Tukey’s test (D, G) and Fisher’s test (E, F). In vivo studies were performed in duplicate; n=10 mice per group. Scale bars, 100 µm in (C). ICC, intrahepatic cholangiocarcinoma; IHC, immunohistochemistry; PlGF, placental growth factor.
Figure 6
Figure 6
PlGF inhibition reduces ICC desmoplasia and improves tumour blood perfusion by opening collapsed vessels. (A) PlGF inhibition induced opening of collapsed blood vessels (representative IHC of vessels in inset). (B) Representative immunofluorescence (IF) for collagen I and CK19 (upper panel) and α-SMA and CK19 (lower panel) in tumours after PlGF inhibition. (C–E) Quantification of IF data showing that PlGF blockade and PlGF knockdown in ICC cells reduced CK19-positive cell area (C) and collagen I expression (D); α-SMA+ ‘myofibroblast-like’ CAF density was reduced in the PlGF knockdown groups, most substantially after PlGF blockade (E). (F) PlGF blockade significantly decreased tumour stiffness. (G) Representative OCT of patent blood vessels in ICC with or without PlGF blockade. (H) Anti-PlGF treatment significantly reduced intratumour hypoxia, reflected by reduced hif1 transcription. P values from Tukey’s test (A, C–E) and Mann-Whitney U teat (F, H). Scale bars, 500 µm in (C). ANOVA, analysis of variance; ICC, intrahepatic cholangiocarcinoma; IHC, immunohistochemistry; OCT, optical coherence tomography; PlGF, placental growth factor.
Figure 7
Figure 7
PlGF blockade enhances the anti-tumour effects of conventional chemotherapy and increases survival. (A, B) The combination of anti-PlGF antibody 5D11D4 (5D11) with conventional gemcitabine plus cisplatin (GC) chemotherapy for 3 weeks significantly delayed tumour growth compared with GC or 5D11 alone (individual growth curves in A) and average tumour size with SD in surviving mice in B). (B) Kaplan-Meier survival distributions after continuous treatment with GC, 5D11 or their combination vs control in mice with established ICC. In vivo studies were performed in duplicate; n=10 mice per group. (D) Addition of 5D11 did not further reduce the viability of 425 cells after gemcitabine (Gem) treatment in vitro. P values from Mann-Whitney U test (B) and log-rank test (D). (E) Proposed mechanism of action of PlGF blockade in ICC: PlGF is produced primarily by CAFs and affects both cancer and stromal cells in an autocrine and paracrine manner in ICC. PlGF inhibition reduces ICC cell invasion and tumour desmoplasia and stiffness, which enhances blood perfusion and the efficacy of chemotherapy. CAF, cancer-associated fibroblast; ICC, intrahepatic cholangiocarcinoma; PlGF, placental growth factor.
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