Journal: Nature Communications

Article Title: Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41467-023-36521-0

Figure Lengend Snippet: a Recognition of the coadaptation process between disseminated tumour cells (DTCs) and hepatocytes in liver metastasis of PDAC. b Brief procedure displaying the intrasplenic injection model for the liver metastasis process study. ( c ) Liver metastasis model by intrasplenic injection of murine Kpc1199 luc cells followed by CT combined with 3D organ reconstruction bioluminescence imaging to assess the time point. Mice that received an intrasplenic injection were photographed to detect the normal liver (NL), (Day 3, D3), premetastatic niche (PMN), (Day 8, D8) and macrometastatic niche (MMN) (Day 15, MMN). Red, reconstructed liver; orange, reconstructed spleen; yellow, signals of tumour niches. Reconstructed organ opacity, left, 100%; right, 20%. Scale colour bar: 2.00 × 10 5 −2.00 × 10 7 ( n = 5 mice per group). d Representative livers from liver metastasis mouse models showing an NL (D3), PMN (D8) or MMN (D15) after injection ( n = 5 mice per group). e IHC-P staining of GFP in left lobules from mouse models showing an NL (D3), PMN (D8) or MMN (D15) ( n = 5 samples per group, 3 fields assessed per sample). Scale bars, 100 μm. f Representative IHC-P staining of the PMN-specific secreted protein markers LOX, SAA1/2, MIF or TIMP1 in the left lobules of livers from model mice to detect an NL (D3), PMN (D8) or MMN (D15) ( n = 5 samples per group, 3 fields assessed per sample). Scale bars, 100 μm; * represents metastatic niches. g, h Representative IHC-P staining of PMNs expressing the tumour-associated macrophage (TAM) markers CD68, CD11b, CD163 and Ly-6G in the left lobules of livers from mouse models to detect an NL (D3), PMN (D8) or MMN (D15) ( n = 5 samples per group, 3 fields assessed per sample). Scale bars, 100 μm; * represents metastatic niches. ( n = 5 samples per group, mean ± SEM, two-tailed unpaired t test, 3 fields assessed per sample). i Representative IHC-P staining of the PMN-specific secreted protein marker LOX and TAM markers CD68 and CD163 in the left lobules of livers from model mice on D8 bearing sham, LTPA modelling and Kpc1199 modelling ( n = 5 samples per group, 3 fields assessed per sample). Scale bars, 100 μm. Source data are provided in the Source Data file.

Article Snippet: Human PDAC cell lines PANC-1, BxPC-3, CFPAC-1, HPAC, CAPAN-1, CAPAN-2, Patu 8988, MIA PaCa-2, SW-1990 and AsPC-1 were purchased from ATCC; Murine cell lines Panc02, Kpc1199 and LTPA were gifting from Professor Jing Xue (State Key Laboratory of Oncogenes andRelated Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University).

Techniques: Injection, Imaging, Paraffin-embedded Immunohistochemistry, Staining, Expressing, Two Tailed Test, Marker

Journal: Nature Communications

Article Title: Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41467-023-36521-0

Figure Lengend Snippet: a , b Livers from intrasplenic injection mouse models induced with Kpc1199 ( a ) or Panc02 ( b ) cells were subjected to transcriptional analysis after confirmation of premetastatic niche (PMN) markers (e.g., CD163, etc.) ( n = 3 mice per group, 5 fields assessed per sample). Scale bars, 500 μm for H&E staining or 100 μm for IHC-P; * represents metastatic niches. c Gene set enrichment analysis (GSEA) based on the gene expression profiles of liver tissues undergoing different metastatic processes after mouse modelling with Panc02 or Kpc1199 cells (false discovery rate analysis; NES, normalized enrichment score). d Venn diagram displaying secreted axon guidance genes upregulated in both PMNs and macrometastatic niche (MMN) compared to levels in normal liver (NL) Panc02 and Kpc1199 cells based on the gene expression profiles of liver tissues from mouse models. e Real-time PCR showing the relative mRNA level of SLIT2 in the left lobule of livers from Kpc1199 cell-injected mouse models on D3, D7, D10 or D15 ( n = 5 per group, mean ± SEM.; two-tailed unpaired t test). ( f ) Representative IHC-P staining of H&E, GFP or SLIT2 in the left lobule of livers from Kpc1199 cell-injected mouse models on D3 (NL), D8 (PMN), D15 or D20 (MMN) ( n = 5 samples per group, 3 fields assessed per sample); * represents metastatic niches. Scale bars, 100 μm. g, h Representative H&E or IHC-P staining of SLIT2 in PMNs ( g ) or MMNs ( h ) formed in the left lobule of livers from Kras G12D/+ / Trp53 R172H/+ / Pdx1 -Cre (KPC) mice bearing spontaneous PDAC liver metastasis ( n = 6 samples per group, 3 fields assessed per sample). Scale bars, 100 μm. Source data are provided in the Source Data file.

Article Snippet: Human PDAC cell lines PANC-1, BxPC-3, CFPAC-1, HPAC, CAPAN-1, CAPAN-2, Patu 8988, MIA PaCa-2, SW-1990 and AsPC-1 were purchased from ATCC; Murine cell lines Panc02, Kpc1199 and LTPA were gifting from Professor Jing Xue (State Key Laboratory of Oncogenes andRelated Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University).

Techniques: Injection, Staining, Paraffin-embedded Immunohistochemistry, Gene Expression, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Nature Communications

Article Title: Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41467-023-36521-0

Figure Lengend Snippet: a Representative IF staining showing the location of SLIT2 and CK19 in adjacent liver or liver metastases of PDAC patients. SLIT2, green; CK19, red; DAPI, blue ( n = 35 patients, 3 fields assessed per sample). Scale bar, 100 μm. b Representative IHC-P staining of CK19 and SLIT2 in liver metastatic niches in PDAC patients ( n = 35 cases, 3 fields assessed per sample). Scale bars, 100 μm. c, d Representative IF staining showing the location of SLIT2 and albumin in the PMN in the Kras G12D/+ / Trp53 R172H/+ / Pdx1 -Cre (KPC) mouse model ( c ) or adjacent liver of metastatic niches in PDAC patients ( d ). Albumin, green; SLIT2, red; DAPI, blue ( n = 35 samples for patients, n = 6 samples per group for mouse models, 3 fields assessed per sample). Scale bars, 50 μm. e Representative IF staining showing the location of ROBO1 and CK19 in the liver metastasis of PDAC patients. ROBO1, green; CK19, red; DAPI, blue ( n = 35 samples, 3 fields assessed per sample). Scale bar, 50 μm. f Representative RNAscope staining displaying the location of SLIT2 mRNA and ALB (upper), KRT19 (middle) and ACTA1 (bottom) mRNAs in the liver metastasis (LM) of PDAC patients. ALB , KRT19 or ACTA1 , green; SLIT2, red; DAPI, blue ( n = 5 samples, 3 fields assessed per sample). Scale bar, 100 μm. g Representative RNAscope staining displaying the location of Slit2 mRNA and Alb mRNA in the premetastatic niche (PMN) and macrometastatic niche (MMN) of Kpc1199 model mice. Alb , green; Slit2 , red; DAPI, blue ( n = 5 samples, 3 fields assessed per sample). Scale bar, 100 μm. h Representative IF staining displaying the expression of ROBO1 in metastatic livers with or without SLIT2 enrichment. CK19, green; ROBO1, red; DAPI, blue ( n = 6 mice per group, 3 fields assessed per sample). Scale bar, 500 μm. Source data are provided in the Source Data file.

Article Snippet: Human PDAC cell lines PANC-1, BxPC-3, CFPAC-1, HPAC, CAPAN-1, CAPAN-2, Patu 8988, MIA PaCa-2, SW-1990 and AsPC-1 were purchased from ATCC; Murine cell lines Panc02, Kpc1199 and LTPA were gifting from Professor Jing Xue (State Key Laboratory of Oncogenes andRelated Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University).

Techniques: Staining, Paraffin-embedded Immunohistochemistry, RNAscope, Expressing

Journal: Nature Communications

Article Title: Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41467-023-36521-0

Figure Lengend Snippet: a Representative H&E staining and IHC-P staining of ROBO1 in human PDAC primary tumours and paired liver metastatic niches displaying the importance of ROBO1 in liver metastasis ( n = 35 cases, 3 fields assessed per sample). Scale bars, 100 μm. b The number of PDAC primary tumours or liver metastases expressing different ROBO1 levels according to scores on IHC-P staining is shown ( n = 35 cases). c Equal amounts of Panc02 Ctrl and Panc02 Robo1 -FL/GFP cells were mixed to prepare an intrasplenic injection model followed by metastatic niche separation and reculture. IHC-P and flow cytometry were performed to evaluate coadaptation. PG0: original cell mixture; PG1: cells derived from PG0-modelled liver metastases; PG2: cells derived from PG1-modelled liver metastases. d–e Flow cytometry to detect the cell composition of PG0, PG1 or PG2 ( n = 5 technical repeats per group). f Heatmap showing the distribution of ROBO1 and SLIT2 expression in the livers of Panc02 cell mixture-modelled mice measured by IHC-P staining ( n = 32 mice per group, 3 fields assessed per sample; independent experiments for each group). N: negative; W, weak; M, moderate; P, positive. g, h Mice treated with ROBO1 neutralizing antibody were modelled with a Panc02 cell mixture before IHC-P staining was performed ( n = 32 mice per group, 3 fields assessed per sample; independent experiments for each group). N: negative; W, weak; M, moderate; P, positive. i The strategy for exploring the coadaptation mechanism mediated by SLIT2-ROBO1 in liver metastatic niches. A mixture of equal amounts of Kpc1199 Robo1 -FL/GFP cells and Kpc1199 ΔRobo1/mCherry cells (Mix-I) or Kpc1199 Robo1 -FL/GFP cells and Kpc1199 CTRL /mCherry cells (Mix-II) was intrasplenically injected into Slit2 /CKO or CTRL mice for further examination. j, k Representative IF staining of a Kpc1199 cell mixture showing the formation of liver metastatic niches indicating coadaptation ( n = 5 mice per group, 3 fields assessed per sample, mean ± SEM.; two-tailed unpaired t test). GFP, green; mCherry, red; DAPI, blue. Scale bars, 100 μm. l Flow cytometry detection of the ratio of two types of Kpc1199 cells in separated liver metastatic niches. Source data are provided in the Source Data file.

Article Snippet: Human PDAC cell lines PANC-1, BxPC-3, CFPAC-1, HPAC, CAPAN-1, CAPAN-2, Patu 8988, MIA PaCa-2, SW-1990 and AsPC-1 were purchased from ATCC; Murine cell lines Panc02, Kpc1199 and LTPA were gifting from Professor Jing Xue (State Key Laboratory of Oncogenes andRelated Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University).

Techniques: Staining, Paraffin-embedded Immunohistochemistry, Expressing, Injection, Flow Cytometry, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41467-023-36521-0

Figure Lengend Snippet: ( a ) Summary model displaying the process of SLIT2-ROBO1-mediated coadaptation of hepatic cells and tumour cells, which promotes metastatic niche outgrowth in PDAC. b Summary model showing the dual roles of ROBO1 in coadaptation with or without SLIT2. ROBO1 induces apoptosis through the caspase cascade in the absence of SLIT2 and triggers proliferation and survival through the p38MAPK pathway by interacting with SLIT2.

Article Snippet: Human PDAC cell lines PANC-1, BxPC-3, CFPAC-1, HPAC, CAPAN-1, CAPAN-2, Patu 8988, MIA PaCa-2, SW-1990 and AsPC-1 were purchased from ATCC; Murine cell lines Panc02, Kpc1199 and LTPA were gifting from Professor Jing Xue (State Key Laboratory of Oncogenes andRelated Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University).

Techniques:

Journal: Scientific Reports

Article Title: A Tumor-Selective Monoclonal Antibody from Immunization with a Tumor-Associated Mucin Glycopeptide

doi: 10.1038/s41598-019-42076-2

Figure Lengend Snippet: Antigen/vaccine structures and conjugation procedures used in this work. ( A ) structure of the 16-residue tandem repeat peptide from MUC4 with a TF ag at position Ser5 (5TF ag -TR MUC4 ) and the PEG linker used to conjugate to gold nanoparticles to construct our vaccine platform (shown), as well as to prepare KLH and BSA conjugates ( B ).

Article Snippet: All FFPE materials, including tissue section, TMA, and cell pellet slides, were stained with the same methods, with anti-MUC4 antibody (Rockland 600-401-GY2, Rabbit, 1:1000).

Techniques: Conjugation Assay, Residue, Construct

Journal: Scientific Reports

Article Title: A Tumor-Selective Monoclonal Antibody from Immunization with a Tumor-Associated Mucin Glycopeptide

doi: 10.1038/s41598-019-42076-2

Figure Lengend Snippet: Flow cytometry histograms of pAb binding to MUC4 + and MUC4 − cell lines.

Article Snippet: All FFPE materials, including tissue section, TMA, and cell pellet slides, were stained with the same methods, with anti-MUC4 antibody (Rockland 600-401-GY2, Rabbit, 1:1000).

Techniques: Flow Cytometry, Binding Assay

Journal: Scientific Reports

Article Title: A Tumor-Selective Monoclonal Antibody from Immunization with a Tumor-Associated Mucin Glycopeptide

doi: 10.1038/s41598-019-42076-2

Figure Lengend Snippet: Plot of signal intensity relative to glycan families for pAb (1:1000 dilution) binding the glycan microarray. Only the MUC4 peptides/glycopeptides that printed bound to the serum, while no other TF-containing molecule bound.

Article Snippet: All FFPE materials, including tissue section, TMA, and cell pellet slides, were stained with the same methods, with anti-MUC4 antibody (Rockland 600-401-GY2, Rabbit, 1:1000).

Techniques: Glycoproteomics, Binding Assay, Microarray

Journal: Scientific Reports

Article Title: A Tumor-Selective Monoclonal Antibody from Immunization with a Tumor-Associated Mucin Glycopeptide

doi: 10.1038/s41598-019-42076-2

Figure Lengend Snippet: Staining of various MUC4 + and MUC4 − cell pellets with affinity-purified pAb serum. Some non-specific, acellular staining is seen within MiaPACA2 (MUC4 − ) cells. No staining was observed for control liver and PANC1 (MUC4 − ) cell pellets while HPAFII and HPAC pellets that are known to express large amounts of MUC4 were strongly and diffusely positive.

Article Snippet: All FFPE materials, including tissue section, TMA, and cell pellet slides, were stained with the same methods, with anti-MUC4 antibody (Rockland 600-401-GY2, Rabbit, 1:1000).

Techniques: Staining, Affinity Purification, Control

Journal: Scientific Reports

Article Title: A Tumor-Selective Monoclonal Antibody from Immunization with a Tumor-Associated Mucin Glycopeptide

doi: 10.1038/s41598-019-42076-2

Figure Lengend Snippet: A section of pancreatic adenocarcinoma with vascular invasion is evaluated for MUC4 expression. The primary tumor mass ( A ) is heterogenous for MUC4 expression, with some neoplastic cells demonstrating strong positive staining while others are negative. At higher power, MUC4 staining is intense within some neoplastic cells ( B ) and not expressed in others. Numerous tumor emboli are present within lymphatic vessels in this tissue section; essentially all neoplastic cells invading vascular structures are positive for MUC4, suggesting the antiserum recognizes tumor clones more likely to produce metastasis within the heterogenous neoplastic tissue.

Article Snippet: All FFPE materials, including tissue section, TMA, and cell pellet slides, were stained with the same methods, with anti-MUC4 antibody (Rockland 600-401-GY2, Rabbit, 1:1000).

Techniques: Expressing, Staining, Clone Assay