The expression of 43 collagen genes, LAIR-1 and LAIR-2 in normal (gray) and tumor tissue (red) were queried using TCGA database. (A) The expression of 43 different collagen genes was assessed …
The expression of total collagens (43 collagen chains), LAIR-1 and LAIR-2 in normal (gray) and tumor tissue (red) was queried using TCGA database: bladder urothelial carcinoma (BLCA), breast …
(A) LAIR-2 overexpression is associated with improved overall survival in some tumors: head and neck squamous cell carcinoma (HNSC), thyroid carcinoma (THCA), thymoma (THYM) and skin cutaneous …
NC410 dose-response measured by Incucyte imaging during 24 hr. Representative microscopic images (10×) of WT and hLAIR-1 reporter cells over time. NC410 protein was added at increasing …
In a non-tumor model, 1 × 107 total human peripheral blood mononuclear cells were adoptively transferred intravenously to NSG mice (N = 6/group) on day 0. Mice were treated with indicated doses of …
Human peripheral blood mononuclear cells were transferred into NSG mice, and at day 6 post transfer human T cells subsets were analyzed in the spleen based on CD45RA and CCR7 expression (naïve …
For collagen gene expression analysis of HT-29 cells, data was acquired from the public dataset GSE41586 (https://pubmed.ncbi.nlm.nih.gov/23902433/). Raw count data of untreated HT-29 cells was …
Immunofluorescence analysis of HT-29 cells stained with (A) isotype control, (B) NC410 or (C) pan-collagen antibody. 40× magnification (D) HT-29 cells were removed from culture flasks using …
(A) Humanized tumor model (A) of HT-29 tumor injected subcutaneously in the presence of human peripheral blood mononuclear cells (PBMCs). 1 × 107 total human PBMCs with or without T cell depletion …
(A) Histogram plot showing mouse P815 tumor cells stained with NC410-biotin or control Fc-biotin. (B) Mouse P815 cells and human HT-29 cells were stained with titrated concentrations of …
(A) Humanized tumor model of HT-29 tumor cells injected subcutaneously in the presence of human peripheral blood mononuclear cells (PBMCs). 2 × 107 total human PBMCs were adoptively transferred …
(A) Schematic representation of the humanized murine tumor model used. HT-29 tumor was injected subcutaneously in the presence of human peripheral blood mononuclear cells. Mice were treated with …
(A) Schematic representation of the immunohistochemical stainings performed. (B) Representative hematoxylin and eosin (H&E), Masson Trichrome, NC410 and LAIR-1 staining in a stomach tumor specimen. …
(A) Representative NC410, LAIR-1, CD45, CD3, CD68 and CD163 staining in a stomach tumor specimen. For quantification of the immune cell counts, regions of interest (ROIs) were annotated on the …
Representative hematoxylin and eosin (H&E), Masson Trichrome, NC410 and LAIR-1 staining for seven different tumor types (glioblastoma [GBM], head and neck squamous cell carcinoma [HNSC], skin …
Representative hematoxylin and eosin (H&E), Masson Trichrome, NC410 and LAIR-1 staining for healthy tissue matching the tumors used in Figure 7—figure supplement 2.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Genetic reagent (Mus musculus) | NOD-SCID IL2Rγnull | Jackson Labs | Stock #: 005557, RRID:IMSR_JAX:005557 | Female mice |
Biological sample (Homo sapiens) | Human PBMCs | In vitro studies – healthy donors in agreement with ethical committee of the University Medical Center Utrecht In vivo studies – Leukopaks (StemCell) | ||
Cell line (M. musculus) | hLAIR-1 reporter cell line | Meyaard Lab | 2B4 T cell hybridoma cells transduced with a NFAT-GFP reporter and hLAIR-1-CD3ζ | |
Cell line (M. musculus) | WT reporter cell line | Meyaard Lab | 2B4 T cell hybridoma cells transduced with a NFAT-GFP reporter | |
Cell line (Homo sapiens) | HT-29 | ATCC | ATCC HTB-38 (RRID:CVCL_0320) | |
Cell line (M. musculus) | P815 | ATCC | ATCC TIB-64 (RRID:CVCL_2154) | |
Cell line (Cricetulus griseus) | CHO cells | Lonza | CHOK1SV, RRID:CVCL_DR97 | CHOK1-SV-GS (glutamine synthase)-KO cells |
Antibody | Anti-human CD45-BV421 (mouse monoclonal) | ThermoFisher Scientific | Cat number: 50-166-070 | (Flow cytometry: 1:40) |
Antibody | Anti-mouse CD45-APC (rat monoclonal) | ThermoFisher Scientific | Cat number: 17-0451-82 | (Flow cytometry: 1:200) |
Antibody | Anti-human CD3-PerCP.Cy5.5 (mouse monoclonal) | ThermoFisher Scientific | Cat number: 45-0037-42 | (Flow cytometry: 1:40) |
Antibody | Anti-human CD8-AF488 (rat monoclonal) | ThermoFisher Scientific | Cat number: 53-0081-82 | (Flow cytometry: 1:40) |
Antibody | Anti-human CD4-BV711 (mouse monoclonal) | Biolegend | Cat number: 300558 | (Flow cytometry: 1:40) |
Antibody | Anti-Human CD45RA- eF450 (mouse monoclonal) | ThermoFisher | Cat number: 48-0458-42 | (Flow cytometry: 1:40) |
Antibody | Anti-human CCR7-PE (mouse monoclonal) | Biolegend | Cat number: 353204 | (Flow cytometry: 1:40) |
Antibody | Anti-human TNF-α-PE (mouse monoclonal) | ThermoFisher Scientific | Cat number: 12-7349-41 | (Flow cytometry: 1:40) |
Antibody | Anti-human IFN-ɣ-PECy7 (rat monoclonal) | ThermoFisher Scientific | Cat number: 25-7311-41 | (Flow cytometry: 1:40) |
Antibody | Anti-human LAIR-1 (rabbit polyclonal) | ATLAS antibodies | Cat number: HPA011155 | (Immunohistochemistry: 1:500) |
Antibody | Anti-human LAIR-1 (mouse monoclonal) | BD Pharmingen | Cat number: 550810 | (Immunohistochemistry: 1:100) |
Antibody | NC410-biotin | This paper/NextCure | See Materials and methods (Immunohistochemistry: 1:500) | |
Antibody | Anti-human CD45 (mouse monoclonal) | DAKO | Cat number: GA751 | (Immunohistochemistry: 1:25) |
Antibody | Anti-human CD3 (rabbit polyclonal) | DAKO | Cat number: GA503 | (Immunohistochemistry: 1:100) |
Antibody | Anti-human CD68 (mouse monoclonal) | Novocastra | Cat number: NCL-L-CD68 | (Immunohistochemistry: 1:1600) |
Antibody | Anti-human CD163 (mouse monoclonal) | Novocastra | Cat number: NCL-L-CD163 | (Immunohistochemistry: 1:800) |
Antibody | Pan-collagen antibody (rabbit polyclonal) | ThermoFisher Scientific | Cat number: PA1-85324 | (Immunofluorescence: 1:200) |
Antibody | Streptavidin APC | eBioscience | Cat number: 17-4317-82 | (Flow cytometry: 1:200) |
Antibody | IgG1-AlexaFluor 594 | ThermoFisher Scientific | Cat number: A-21125 | (Immunofluorescence: 1:200) |
Antibody | Streptavidin AlexaFluor 594 | ThermoFisher Scientific | Cat number: S11227 | (Immunofluorescence: 1:2000) |
Peptide, recombinant protein | LAIR-1 FC | This paper/NextCure | See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation | |
Peptide, recombinant protein | LAIR-2 FC | This paper/NextCure | See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation | |
Peptide, recombinant protein | LAIR-2 FES | This paper/NextCure | See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation | |
Peptide, recombinant protein | Human collagen I | R&D Systems; Stem Cell | Cat number: 6220 CL-020 (R&D); 07005 (StemCell) | |
Peptide, recombinant protein | Mouse collagen I | Ray Biotech | Cat number: DF-01-0058 | |
Peptide, recombinant protein | Rat collagen I | Yo Protein | Cat number: ABIN628947 | |
Peptide, recombinant protein | Human collagen III | R&D Systems | Cat number: 7294 CL-020 | |
Peptide, recombinant protein | Rat collagen III | Yo Protein | Cat number: ABIN377054 | |
Commercial assay/kit | mouse tumor dissociation kit | Miltenyi | Cat number: 130-096-730, RRID:SCR_020285 | |
Commercial assay/kit | Cell Stimulation Cocktail plus protein transport inhibitors | ThermoFisher Scientific | Cat number: 00-4970-93 | |
Commercial assay/kit | Cytofix/CytoPerm Plus Fixation/Permeabilization Kit | BD | Cat number: 555028 | |
Commercial assay/kit | Singleplex Luminex Protein Assay Kit | ThermoFisher Scientific | Cat number: EPX010-10420-901 | |
Commercial assay/kit | Human T cell positive selection kit | StemCell | Cat number: 17851 | |
Commercial assay/kit | Optiview DAB IHC detection kit | Ventana kit | Cat number: 760–700 | |
Other | Zombie NIR viability dye | Biolegend | Cat number: 423105 | |
Other | ACK lysis buffer | KD medical | Cat number: 50-101-9080 | |
Other | RIPA Lysis buffer | ThermoFisher Scientific | Cat number: 89900 | |
Other | proteinase inhibitor | Roche | Cat number: 4693116001 | |
Other | DNase | Millipore | Cat number: 69182–3 | |
Other | Collagenase (from Clostridium histolyticum) | Sigma | Cat number: C0130 | |
Other | Triton X-100 | Roche | Cat number: 10789704001 | |
Other | EZ-Link NHS-PEG4-Biotin | ThermoFisher | Cat number: 21329 | |
Other | ZebaSpin Desalting Columns | ThermoFisher | Cat number: 87770 | |
Other | DELFIA wash buffer | PerkinElmer | Cat number: 1244-114 | |
Other | DELFIA enhancement solution | PerkinElmer | Cat number: 4001-0010 | |
Other | Europium-labeled Streptavidin | PerkinElmer | Cat number: 1244-360 | |
Other | Masson’s Trichrome | Abcam | Cat number: Ab150686 | |
Other | Hematoxylin | Sigma-Aldrich chemie | Cat number: 51275-1L | |
Other | DAPI VectaShield hardset | Vector Lab | Cat number: H-1500-10, RRID:AB_2336788 | |
Software, algorithm | QuPath version 0.2.0 | QuPath | RRID:SCR_018257 | |
Software, algorithm | GraphPad Prism 8.0 | GraphPad Prism | RRID:SCR_002798 | |
Software, algorithm | TCGA database (https://cancergenome.nih.gov/) | RRID:SCR_003193 | R codes for TCGA analysis Files can be found in the supplied Source code files |
Survival curves comparing individuals with high collagen expression against individuals with low collagen expression.
Expression values corresponding to 43 collagen genes in tumor were obtained and averaged for each individual. The collagen expression from various individuals was divided into four quantiles. The individuals with expression values less than or equal to the first quantile were regarded as low expression, and those with values greater than or equal to the third quantile were considered as high expression. The association between expression (high/low) and their survival was assessed using Kaplan–Meier method. The survival curves were drawn using ggsurvplot function in the survminer package in R. The cancers where p-value was less than 0.05 were considered significant and are displayed.
Average collagen expression in normal and tumor across various cancers.
The expression values corresponding to 43 different collagen proteins are queried from the TCGA database. TCGA consists of 33 projects corresponding to 33 different cancers, which can individually be queried for expression of genes. For each cancer, expression of collagen genes in cancer and normal (where available) was queried individually. The expression across all collagens was averaged in tumor and normal and displayed as a bar graph in different cancers.
Average LAIR-1/2 expression in normal and tumor across various cancers.
Expression of LAIR-1/2 is queried from the TCGA database. Expression of LAIR-1/2 in normal and tumor is displayed as bar graphs in each cancer.
Survival curves comparing individuals with high LAIR-1/2 expression against individuals with low LAIR-1/2 expression.
LAIR-1/2 expression was obtained for each individual and divided into four quantiles. The individuals with expression values less than or equal to the first quantile were regarded as low expression, and those with values greater than or equal to the third quantile were considered as high expression. The association between expression (high/low) and their survival was assessed using Kaplan–Meier method, and the cancers where p-value was less than 0.05 are displayed.
Survival curves comparing individuals with high collagen, high LAIR-1 expression against individuals with low collagen, low LAIR-1 expression.
Two-way analysis involved dividing average collagen expression across individuals into low and high categories based on the quantiles as Source code 4. Similarly, LAIR-1 expression was also divided into low and high categories based on quantiles. The individuals with high LAIR-1 and high collagen expression were considered high group, and those with low LAIR-1 and low collagen expression were considered low group. Survival based on expression of these two categories was evaluated using Kaplan–Meier method, and the cancers where p-value was less than 0.05 is displayed.