Targeting MFAP5 in cancer-associated fibroblasts sensitizes pancreatic cancer to PD-L1-based immunochemotherapy via remodeling the matrix

Highly desmoplastic and immunosuppressive tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) contributes to tumor progression and resistance to current therapies. Clues targeting the notorious stromal environment have offered hope for improving therapeutic response whereas the underlying mechanism remains unclear. Here, we find that prognostic microfibril associated protein 5 (MFAP5) is involved in activation of cancer-associated fibroblasts (CAFs). Inhibition of MFAP5highCAFs shows synergistic effect with gemcitabine-based chemotherapy and PD-L1-based immunotherapy. Mechanistically, MFAP5 deficiency in CAFs downregulates HAS2 and CXCL10 via MFAP5/RCN2/ERK/STAT1 axis, leading to angiogenesis, hyaluronic acid (HA) and collagens deposition reduction, cytotoxic T cells infiltration, and tumor cells apoptosis. Additionally, in vivo blockade of CXCL10 with AMG487 could partially reverse the pro-tumor effect from MFAP5 overexpression in CAFs and synergize with anti-PD-L1 antibody to enhance the immunotherapeutic effect. Therefore, targeting MFAP5highCAFs might be a potential adjuvant therapy to enhance the immunochemotherapy effect in PDAC via remodeling the desmoplastic and immunosuppressive microenvironment.

in low serum medium. Besides, migration and invasion (polycarbonate membrane were coated with Marigel (Corning, #354234) assays were performed in the 24-well transwell systems (8μm pores, Corning). Proper number (5-10×10 4 ) of cells were seeded in the upper insets with serum-free medium whereas complete medium containing 20% FBS was added in the lower well. Cells that went through the membrane were observed and counted under microscope (Leica) after fixation and staining with crystal violet (Sigma-Aldrich).

Three-dimensional (3D) spheroids formation assay, coculture and 3D culture system
In the 3D spheroids cultivation system, hanging drop method was used to form spheroids containing MFAP5_KD or control CAFs, as described elsewhere. [16] CAFs were suspended to a proper concentration and cellular drops (20μl) were laid on the lid of culture plates. PBS was added to the plate for humidity and the lid was inverted allowing growth of the spheroids. Additionally, for 3D heterospheroids containing CAFs and PDAC cells, low-adhesion round-bottom 96-well plates were used and a 1:1 mixture of CAF subpopulations and KPC/Panc02 was seeded and incubated. Gemcitabine (10μM) and vehicle were added to the formed spheroids on the third day and refreshed on the sixth day. For both spheroid systems, morphology and diameter of spheroids was observed and imaged under a microscope. Viability of spheroids were detected with CellTiter-Lumi™ Steady Plus Kit (Beyotime) following the manufacturer's protocol. As for the coculture system, conditioned medium from MFAP5_NC or KD CAFs (collected from equal amount for equal duration) were added to the candidate cells and the conditioned medium was refreshed every 24 hours.
Additionally, the 3D tube formation assay was performed in 96-well plate. 50ul Matrigel was lined at the bottom of the board and wait 30 minutes until solidification, then 2×10 3 HUVEC were seeded in 100ul Matrigel (diluted by DMEM culture medium, 1:1). Stretch of tubes were then examined under a microscope in 1-3 days and the images were then analyzed by Image J 1.8.0 software.

Nuclear-cytoplasmic separation, immunoprecipitation and Western blot assay
The separation of nuclear and cytoplasmic protein was carried out with Nuclear and Cytoplasmic Extraction Reagent (ThermoFisher SCIENTIFIC) according to the manufacturer's protocol. Immunoprecipitation and western blot assay were performed as previously described.
[40] Information of antibody involved in the present study was listed in Table S3. The protein signals were detected and visualized with the ChemiScopeTouch (Clinx Science Instruments, Shanghai, China), and the intensity of western blot bands was measured by Image J 1.8.0 software.

RNA isolation, qRT-PCR, dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay
Cellular total RNA was extracted with TRIzol reagents (Invitrogen) and qRT-PCR was performed according to standard protocols as previously described.
[40] Chromatin immunoprecipitation (ChIP) assay was performed with ChIP Assay Kit (Beyotime) according to the manufacturer's protocols. The promoter regions containing 3000bp upstream sequence as well as 5'UTR were cloned into the luciferase reporter vector.
Two most predicted binding sites were mutant defined as mut1 and mut2. Relative luciferase activity was measured with dual-luciferase reporter assay system (Promega) according to the manufacturer's protocols. Primer sequences were listed in Table S4.

IHC, chemical staining and Immunofluorescence staining
Immunohistochemistry (IHC) and immunofluorescence staining were performed as previously described (38). In our present study, staining of stromal deposited collagen was performed with Picrosirius Red Stain Kit (Polysciences, INC) and hyaluronic acid staining was performed with Alcian blue -Nuclear solid red staining Kit (Beyotime) following the manufacturer's protocols. Besides, IHC staining of HAS2 was also performed considering the non-specific staining of both collagen and mucins with Alcian blue alone. Notably, most of our findings were verified with more than one method in at least 3 repeated individual experiments, and optimal statistical methods were performed to evaluate the differences. The multiplex immunofluorescence was performed according to the manufacture's protocol via usage of Akoya Biosciences Polaris Kit.  in PDAC tissues from The Human Protein Atlas database. Density of colored points represents the relative expression according to fixed interval of read count (0, 1, 2-4, 5-9, >10). Column diagram is shown on the right. MFAP5 enrichment in single cell sequencing clusters of mouse pancreas tissues in PanglaoDB database. (C) Gene co-expression analysis between MFAP5 and collagen-related genes.   Figure 3B. Scale bars, 50μm, 100μm, 200μm. (E) Representative images of immunofluorescent staining of RFP in C67BL/6 mice. The ImdyCAFs were stably transfected with vector with RFP tag. The data were analyzed by a twotailed unpaired Student's t-test (A, B). Error bars, means ± SD of three independent experiments. **P < 0.01, ns, not significant.  (D) Western analysis of EMT-related proteins of Panc-1 and Panc02 cells in coculture system with or without AMG487 (5nM, 24h). (E) Representative images of 2D/3D tube formation assays of HUVEC cells cocultured in coculture system with or without AMG487(5nM, 24h). Scale bar: 50μm. (F) Schematic procedures of orthotopic co-injection of ImdyCAFs and KPC cells into C57BL/6 mice combined with administration of AMG487 (5mg/kg). (G) Images and statistical tumor weight of isolated tumors derived from Panc02 and MFAPF_OE or control ImdyCAFs with administration of AMG487 (5mg/kg). A total of 15 mice were analyzed (5 mice for each group individually). (H) Representative IHC staining images and histograms of tumors in Figure 5J, and the statistical analysis of Ki67, CD31 and Granzyme B. (I) Roles of AMG487 on tumors generated from KPC cell lines alone. Statistical results of the tumor weight and survival benefit were shown on the right. Scale bar: 50μm. The data were analyzed by a twotailed unpaired Student's t-test (C, E, G, H, I). Error bars, means ± SD of three independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001, ns, not significant.

Figure S8 Additional results of MFAP5 regulates tumoral PD-L1 via CXCL10. (A)
Schematic procedures of orthotopic co-injection of ImdyCAFs and KPC cells into C57BL/6 mice combined with administration of AMG487 (5mg/kg) and anti-PD-L1 antibody (200μg/mouse). (B) Changes of mouse body weight during the experiment. (C) Detection of serum chemical indicators of mice cotreated with AMG487 (5mg/kg) and anti-PD-L1 antibody. Error bars, means ± SD of three independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001, ns, not significant. Schematic procedures of orthotopic co-injection of stable transfected ImdyCAFs and Panc02 or KPC cells into C57BL/6 mice combined with administration of gemcitabine (50mg/kg) every three days. (B) Images and (C) statistical tumor weight of isolated co-injected KPC tumors after mice were sacrificed at day 28. A total of 20 mice were analyzed (5 mice for each group individually). (D) Representative IHC staining images and statistical analysis of Ki67 and Granzyme B. Scale bar: 50μm. The data were analyzed by a two-tailed unpaired Student's t-test (C, D). Error bars, means ± SD of three independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001.