Journal: Nature Communications

Article Title: Cancer cell survival depends on collagen uptake into tumor-associated stroma

doi: 10.1038/s41467-022-34643-5

Figure Lengend Snippet: Orthotopic growth of a EO771 mammary or subcutaneous growth of b Glioma261 tumors in C57BL6 TEM8 WT or KO mice or subcutaneous growth of c HPAC pancreatic tumors in athymic nude TEM8 WT or KO mice. n = 21 (WT) or 19 (KO) (E0771), 8 (WT) or 15 (KO) (Glioma261), and 10 (WT) or 12 (KO) (HPAC) biologically independent animals per group. d Growth of RENCA kidney tumors in BALB/c TEM8 WT or KO mice. n = 14 biologically independent animals per group. e Growth of MC38 tumors in C57BL6 mice containing a TEM8 knockout (KO) allele, or a deletion of the transmembrane domain (Lepp-Del-TM) along with corresponding wildtype controls. n = 28 (TEM8 WT), 24 (TEM8 KO), 28 (Lepp WT), and 25 (Lepp-Del-TM) biologically independent animals per group. *; P < 0.0001 between WT and KO and between Lepp-WT and Lepp-Del-TM at 10-, 13- and 15-days post inoculation. f Spontaneous mammary tumor growth in MMTV-PyMT transgenic TEM8 WT and KO on an FVB background. Total tumor burden was measured weekly until mice reached 13 weeks of age. Tumors developed in 100% of TEM8 WT and 93% of TEM8 KO mice. n = 30 biologically independent animals per group. Growth of MC38 tumors in TEM8 WT or KO mice at 4 ( g ) or 11 months ( h ) of age. TGI; Tumor growth inhibition. n = 12 (4 months) or 15 (11 months) biologically independent animals per group. Data are denoted as mean ± s.e.m. Statistical analysis was calculated using unpaired T tests comparing tumor volume from WT and KO mice (or Lepp-WT and Lepp-Del-TM) at the same day post inoculation. Source data are provided as a file.

Article Snippet: For co- IF staining of vessels and CAFs in tumors, frozen cryosections were fixed with 1% paraformaldehyde (PFA)/PBS and stained with mouse anti-a-Smooth Muscle Actin (SMA) antibody (Sigma cat# A5228) or m825 human anti-TEM8.SMA signal was detected using biotin-donkey anti-mouse IgG (Jackson ImmunoResearch, cat# 715-065-151) secondary antibody followed by Texas red-streptavidin (Vector Laboratories, cat # SA-5006-1) while TEM8 signal was detected using Fluorescein (FITC) goat anti-human IgG (Jackson ImmunoResearch cat# 109-095-088) followed by Alexa-488 goat-anti-FITC (ThermoFisher cat# A-11096).

Techniques: Knock-Out, Transgenic Assay, Inhibition

Journal: Nature Communications

Article Title: Cancer cell survival depends on collagen uptake into tumor-associated stroma

doi: 10.1038/s41467-022-34643-5

Figure Lengend Snippet: Co-immunofluorescence (IF) staining was used to detect CD31 + vasculature (pseudocolored red) and cre (GFP, green) in a B16 tumors from transgenic reporter mice expressing Tie-cre or VE-cad-cre, or b SW620 tumors from SCID transgenic reporter mice expressing Fsp-cre. Bar = 50 μm. Images were representative of three experiments ( n = 3 animals per group). Subcutaneous tumor growth was monitored in VE-cadherin-cre ( c ), Tie2-cre ( d ) or Fsp-cre ( e ) conditional Tem8 KO strains on a C57BL6 (MC38, B16), BALB/c (RENCA) or SCID (UACC, SW620) background. For MC38, n = 14 (TEM8 flox +; VE-cad-Cre+), 11 (TEM8 flox/- ; VE-cad- Cre+), 16 (TEM8 flox +; Tie2-Cre+), 14 (TEM8 flox/- ; Tie2-Cre+), 14 (TEM8 flox +; Fsp-Cre+), and 15 (TEM8 flox/- ; Fsp-Cre+) biologically independent animals per group. For B16, n = 15 (TEM8 flox +; VE-cad-Cre+), 19 (TEM8 flox/- ; VE-cad-Cre+), 13 (TEM8 flox +; Tie2-Cre+) and 13 (TEM8 flox/- ;Tie2-Cre+) biologically independent animals per group. For RENCA, n = 14 (TEM8 flox +; Fsp- Cre+) or 15 (TEM8 flox/- ; Fsp-Cre+) biologically independent animals per group. For UACC, n = 21 (TEM8 flox +; Tie2-Cre+), 17 (TEM8 flox/- ; Tie2-Cre+), 12 (TEM8 flox +; Fsp-Cre+) or 15 (TEM8 flox/- ; Fsp-Cre+) biologically independent animals per group. For SW620 n = 5 (TEM8 flox +; Fsp-Cre+) or 10 (TEM8 flox/- ; Fsp-Cre+) biologically independent animals per group. Data are denoted as mean ± s.e.m. Source data are provided as a file.

Article Snippet: For co- IF staining of vessels and CAFs in tumors, frozen cryosections were fixed with 1% paraformaldehyde (PFA)/PBS and stained with mouse anti-a-Smooth Muscle Actin (SMA) antibody (Sigma cat# A5228) or m825 human anti-TEM8.SMA signal was detected using biotin-donkey anti-mouse IgG (Jackson ImmunoResearch, cat# 715-065-151) secondary antibody followed by Texas red-streptavidin (Vector Laboratories, cat # SA-5006-1) while TEM8 signal was detected using Fluorescein (FITC) goat anti-human IgG (Jackson ImmunoResearch cat# 109-095-088) followed by Alexa-488 goat-anti-FITC (ThermoFisher cat# A-11096).

Techniques: Immunofluorescence, Staining, Transgenic Assay, Expressing

Journal: Nature Communications

Article Title: Cancer cell survival depends on collagen uptake into tumor-associated stroma

doi: 10.1038/s41467-022-34643-5

Figure Lengend Snippet: a Image depicting amino acids in the TEM8 MIDAS motif that coordinate the metal ion (green). b ELISA was used to measure the binding of AP, TEM8-AP, and D150A-AP to col1. PA was included as a positive control. n = 2 (AP, negative control) or 3 (TEM8-AP and TEM8-D150-AP) biologically independent samples per group. Statistical comparison between AP and D150A- AP was performed using an unpaired T test. c ELISA was used to measure the binding of AP and D150A-AP to various ECM molecules. PA was included as a positive control. n = 3 biologically independent samples per group. Statistical comparison between AP and D150A-AP was performed using an unpaired T test. d IF staining was used to detect TEM8 (green) in CHO and CHO-TEM8 cells. Bar = 20 μm. Images were representative of three experiments. e A cell binding assay was used to measure binding of CHO-TEM8 cells to various ECM molecules. n = 6 biologically independent samples per group. Statistical comparisons between CHO and CHO-TEM8 were performed using an unpaired T test. f IF staining was used to detect CHO-mediated degradation of an underlying FITC-col gel (green). Cell nuclei were visualized using DAPI (blue). Bar = 50 μm. Images were representative of three experiments. g IF staining was used to detect collagen uptake after adding soluble FITC collagen (green) to the media. CellMask orange was used to visualize cell membranes (red) and Hoechst 33342 to visualize nuclei (blue). Bar = 20 μm. Images were representative of three experiments. h Western blotting was used to detect changes in TEM8 expression following exposure of CHO-TEM8 cells to various ECM molecules. Wedge: Col1; 10, 25, and 50 μg/mL, ColVI; 1, 10, 25 μg/mL. Images were representative of three experiments. Data in b , c , and e , are denoted as mean ± s.e.m. Source data are provided as a file.

Article Snippet: For co- IF staining of vessels and CAFs in tumors, frozen cryosections were fixed with 1% paraformaldehyde (PFA)/PBS and stained with mouse anti-a-Smooth Muscle Actin (SMA) antibody (Sigma cat# A5228) or m825 human anti-TEM8.SMA signal was detected using biotin-donkey anti-mouse IgG (Jackson ImmunoResearch, cat# 715-065-151) secondary antibody followed by Texas red-streptavidin (Vector Laboratories, cat # SA-5006-1) while TEM8 signal was detected using Fluorescein (FITC) goat anti-human IgG (Jackson ImmunoResearch cat# 109-095-088) followed by Alexa-488 goat-anti-FITC (ThermoFisher cat# A-11096).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Positive Control, Negative Control, Staining, Cell Binding Assay, Western Blot, Expressing

Journal: Nature Communications

Article Title: Cancer cell survival depends on collagen uptake into tumor-associated stroma

doi: 10.1038/s41467-022-34643-5

Figure Lengend Snippet: a Alignment of TEM8 with the known collagen-binding site of integrins. The conserved mutations in this study, including the Glutamine (E) and Histidine (H) found in collagen binding integrins are highlighted (yellow). b Image depicting the conserved TEM8 surface residues, E152 and H154, predicted to contact collagen based on homology with integrin alpha 2. c An ELISA was used to measure the binding of AP, AP-TEM8 (WT) and various AP-TEM8- mutants to col1. n ≥ 8 biologically independent samples per group. Statistical analysis was calculated using one-way analysis of variance with a Tukey’s test. d A FITC release assay was used to measure soluble FITC in the supernatant of CHO cells, CHO cells expressing wildtype TEM8 (WT) or various TEM8 mutants following culture in a FITC-Col gel. n = 4 biologically independent samples per group. P < 0.0001 between CHO- TEM8 WT and each of the CHO-TEM8 mutants. Statistical analysis was calculated by using one-way analysis of variance with a Tukey’s test. e Flow cytometry was used to measure FITC in CHO, CHO-TEM8 (WT), and CHO-TEM8 mutant cells following FITC-Col treatment. f Growth of MC38 in TEM8 +/+ or TEM8 E150V/E150V mice. n = 20 (TEM8 +/+ ) and 14 (TEM8 E150V/E150V ) biologically independent animals per group. Mice used in this study were 4 to 5 months old. Statistical analysis was calculated using unpaired T tests comparing tumor volume from WT and KI mice on the same day post inoculation. *; P < 0.0001, **; P = 0.002, ***; P = 0.009. Data are denoted as mean ± s.e.m. Source data are provided as a file.

Article Snippet: For co- IF staining of vessels and CAFs in tumors, frozen cryosections were fixed with 1% paraformaldehyde (PFA)/PBS and stained with mouse anti-a-Smooth Muscle Actin (SMA) antibody (Sigma cat# A5228) or m825 human anti-TEM8.SMA signal was detected using biotin-donkey anti-mouse IgG (Jackson ImmunoResearch, cat# 715-065-151) secondary antibody followed by Texas red-streptavidin (Vector Laboratories, cat # SA-5006-1) while TEM8 signal was detected using Fluorescein (FITC) goat anti-human IgG (Jackson ImmunoResearch cat# 109-095-088) followed by Alexa-488 goat-anti-FITC (ThermoFisher cat# A-11096).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Release Assay, Expressing, Flow Cytometry, Mutagenesis

Journal: Nature Communications

Article Title: Cancer cell survival depends on collagen uptake into tumor-associated stroma

doi: 10.1038/s41467-022-34643-5

Figure Lengend Snippet: Western blotting was used to detect TEM8 or CMG2 protein expression in HMECs ( a, d ), TIME ( b ), or TSCs ( c, e ) following serum deprivation ( a–c ) or glutamine (Gln) deprivation ( d, e ). β-actin (β-act) was used as a loading control. Images were representative of three experiments. IHC was used to assess TEM8 protein expression in normal adjacent human colon ( f ) or colorectal cancer ( g ). Bar = 100 μm ( f ) or 200 μm ( g ). Images were representative of three experiments. Source data are provided as a file.

Article Snippet: For co- IF staining of vessels and CAFs in tumors, frozen cryosections were fixed with 1% paraformaldehyde (PFA)/PBS and stained with mouse anti-a-Smooth Muscle Actin (SMA) antibody (Sigma cat# A5228) or m825 human anti-TEM8.SMA signal was detected using biotin-donkey anti-mouse IgG (Jackson ImmunoResearch, cat# 715-065-151) secondary antibody followed by Texas red-streptavidin (Vector Laboratories, cat # SA-5006-1) while TEM8 signal was detected using Fluorescein (FITC) goat anti-human IgG (Jackson ImmunoResearch cat# 109-095-088) followed by Alexa-488 goat-anti-FITC (ThermoFisher cat# A-11096).

Techniques: Western Blot, Expressing

Journal: Nature Communications

Article Title: Cancer cell survival depends on collagen uptake into tumor-associated stroma

doi: 10.1038/s41467-022-34643-5

Figure Lengend Snippet: a Depiction of the collagen degradation pathway in TSCs that results in the production of glutamine (Gln), a transportable metabolite that can be exploited by cancer cells. b Viability of TEM8 wildtype (WT) or knockout (KO) TSCs under nutrient starvation in the presence or absence of col1. n = 5 biologically independent samples per group. Statistical analysis was calculated using an unpaired T test. c Viability of SW620 cancer cells under nutrient starvation in the presence or absence of col1. n = 5 biologically independent samples per group. *; p < 0.0001. Statistical analysis was calculated by using one-way analysis of variance with a Tukey’s test. d Relative ATP production in SW620 cells in the presence or absence of glutamine (Gln). n = 8 biologically independent samples per group. *; p < 0.0001. Statistical analysis was calculated by using an unpaired T test. e Viability of SW620 cancer cells under nutrient starvation in co-cultures with TEM8 wildtype or knockout TSCs. n = 5 biologically independent samples per group. *; p < 0.0001. Statistical analysis was calculated by using one-way analysis of variance with a Tukey’s test. Western blotting was used to detect PEPD ( f ), GLUL ( g ) or PRODH ( h ) expression in wildtype TSCs following knockdown (KD) with non-specific control shRNA or gene-specific shRNA. Images were representative of three experiments. Viability of SW620 cancer cells under nutrient starvation upon co-culture with PEPD ( i ), GLUL ( j ), or PRODH ( k ) knock down (KD) TSCs. NS-shRNA: non-specific control shRNA. n = 5 biologically independent samples per group. *; p < 0.0001, **; p < 0.0003. Statistical analysis was calculated by using one-way analysis of variance with a Tukey’s test. l Relative abundance of each of the 20 amino acids in the supernatants of TSC-WT, TSC-KO, or TSC-WT cells following m830 anti-TEM8 antibody treatment. To minimize experimental variability, data from two experiments were combined ( n = 3 per group). *; p < 0.0001. Statistical analysis was calculated by using one-way analysis of variance with a Tukey’s test. m Viability of nutrient-starved SW620 cancer cells in TSC co-cultures following treatment with L-Asparaginase (L-Asp). n = 5 biologically independent samples per group. *; p < 0.0001. Statistical analysis was calculated by using an unpaired T test. Gln; glutamine, Pro; proline, H; high serum, 10% FBS, L; low serum 0.5% FBS. Data are denoted as mean ± s.d. Source data are provided as a file.

Article Snippet: For co- IF staining of vessels and CAFs in tumors, frozen cryosections were fixed with 1% paraformaldehyde (PFA)/PBS and stained with mouse anti-a-Smooth Muscle Actin (SMA) antibody (Sigma cat# A5228) or m825 human anti-TEM8.SMA signal was detected using biotin-donkey anti-mouse IgG (Jackson ImmunoResearch, cat# 715-065-151) secondary antibody followed by Texas red-streptavidin (Vector Laboratories, cat # SA-5006-1) while TEM8 signal was detected using Fluorescein (FITC) goat anti-human IgG (Jackson ImmunoResearch cat# 109-095-088) followed by Alexa-488 goat-anti-FITC (ThermoFisher cat# A-11096).

Techniques: Knock-Out, Western Blot, Expressing, shRNA, Co-Culture Assay

Journal: Nature Communications

Article Title: Cancer cell survival depends on collagen uptake into tumor-associated stroma

doi: 10.1038/s41467-022-34643-5

Figure Lengend Snippet: a ELISA was used to measure the binding of AP (control) and TEM8-AP to col1 in the absence or presence of 20 μg/mL of m830 anti-TEM8 antibodies. n = 11 biologically independent samples per group. Statistical analysis was calculated by using an unpaired T test. b Flow cytometry was used to quantify FITC-Col uptake in CHO and CHO-TEM8 cells that were treated with non-specific control IgG (IgG) or TEM8 antibodies m830 or SB5. The SB5 anit-TEM8 antibody is unable to block collagen binding and was used as an additional negative control. c Growth of UACC melanoma tumors following treatment with 15 mg/kg of m830 antibody. Treatments were administered 3× per week and initiated (arrow) when tumors reached a size of 60 mm 3 . n = 18 (vehicle) or 12 (m830) biologically independent animals per group. *; P ≤ 0.0001, **; P = 0.0002. Statistical analysis was calculated using unpaired T tests comparing tumor volume from vehicle and m830 treated mice on the same day post inoculation. d Average body weights of mice in c at treatment start (day 6) and study end (day 28). N.S.: non-significant. n = 15 (vehicle) or 12 (m830) independent samples per group. Statistical analysis was calculated by using an unpaired T test. Liver metastases following intrasplenic injection of HCT-116-luc colon cancer was quantified at 21- and 28-days post inoculation (DPI) ( e ) using BLI. Statistical analysis was calculated by using an unpaired T test. Images from five representative mice/group are shown in f and examples of excised livers with tumor lesions (arrowheads) are shown in g . Kaplan- Meier survival analysis ( h ). In this study, treatments began with 15 mg/kg one day following tumor cell inoculation (arrow) followed by 5 mg/kg 3 times/week for 4 weeks. Log-rank analysis: P < 0.0001, m830 versus vehicle. n = 15/group. i Growth of DLD1 colon tumors following treatment with 15 mg/kg of m830 3× per week, 5 mg/kg bevacizumab (Bev) 2× per week, or a combination of m830 and Bev. Treatments were initiated (arrow) when tumors reached an average size of 50 mm 3 . n = 18 (vehicle), 13 (m830), 13 (Bev), or 14 (m830 + Bev) biologically independent animals per group. Statistical analysis was calculated by using an unpaired T test. j Growth of NCI-H460 lung tumors following treatment with 15 mg/kg of m830 3x per week, 30 mg/kg paclitaxel (PT) (qod × 5), or a combination of m830 and PT. Treatments were initiated (arrow) when tumors reached an average size of 60 mm 3 . n = 10 biologically independent animals per group. Statistical analysis was calculated by using an unpaired T test. k Growth of MC38 colon tumors following treatment with 10 mg/kg of m830, 3.5 mg/kg anti- PD1 antibody (αPD1, clone RMPI), or a combination of m830 and αPD1. Treatments were administered 3× per week and were initiated (arrow) when tumors reached an average size of 80 mm 3 . p = 0.01 with respect to αPD1 alone. n = 12 (vehicle), 10 (m830), 14 (anti-PD1) or 14 (m830 + anti-PD1) biologically independent animals per group. Statistical analysis was calculated by using an unpaired T test. Data in a and d are denoted as mean ± s.d. Data in c, i–k are denoted as mean ± s.e.m. Source data are provided as a file.

Article Snippet: For co- IF staining of vessels and CAFs in tumors, frozen cryosections were fixed with 1% paraformaldehyde (PFA)/PBS and stained with mouse anti-a-Smooth Muscle Actin (SMA) antibody (Sigma cat# A5228) or m825 human anti-TEM8.SMA signal was detected using biotin-donkey anti-mouse IgG (Jackson ImmunoResearch, cat# 715-065-151) secondary antibody followed by Texas red-streptavidin (Vector Laboratories, cat # SA-5006-1) while TEM8 signal was detected using Fluorescein (FITC) goat anti-human IgG (Jackson ImmunoResearch cat# 109-095-088) followed by Alexa-488 goat-anti-FITC (ThermoFisher cat# A-11096).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Flow Cytometry, Blocking Assay, Negative Control, Injection

Journal: Science Progress

Article Title: Expression and clinical significance of TEM8 protein in triple-negative breast cancer

doi: 10.1177/00368504251410665

Figure Lengend Snippet: Expression and distribution of TEM8 in TNBC patient tissues. (A–C) Representative IHC images of TEM8 expression in TNBC tissues and adjacent non-tumor tissues (A: A case with high TEM8 expression. B: A case with low TEM8 expression. C: Adjacent normal tissue. 400×). (D) Distribution of TEM8 protein expression scores in 153 TNBC tissue specimens.

Article Snippet: The TEM8 antibody (Rabbit polyclonal; RRID: AB_2056891; Proteintech, USA; Catalog: 15091-1-AP; Working dilution: 1:200) and CD31 antibody (Mouse monoclonal Clone: WM-59;RRID:AB_2935131;Proteintech,USA;Catalog:CL750-65268;Working dilution:1:500) were procured from Proteintech, while the DAB kit (catalog #ZLI-9018) was sourced from Zhongshan Jinqiao Biotech

Techniques: Expressing

Journal: Science Progress

Article Title: Expression and clinical significance of TEM8 protein in triple-negative breast cancer

doi: 10.1177/00368504251410665

Figure Lengend Snippet: Kaplan-Meier survival curves of 118 TNBC patients stratified by TEM8 expression.

Article Snippet: The TEM8 antibody (Rabbit polyclonal; RRID: AB_2056891; Proteintech, USA; Catalog: 15091-1-AP; Working dilution: 1:200) and CD31 antibody (Mouse monoclonal Clone: WM-59;RRID:AB_2935131;Proteintech,USA;Catalog:CL750-65268;Working dilution:1:500) were procured from Proteintech, while the DAB kit (catalog #ZLI-9018) was sourced from Zhongshan Jinqiao Biotech

Techniques: Expressing

Journal: The Journal of Infectious Diseases

Article Title: Anthrax Lethal and Edema Toxins Fail to Directly Impair Human Platelet Function

doi: 10.1093/infdis/jir763

Figure Lengend Snippet: Assessment of anthrax toxin binding to human platelet and platelet surface expression of anthrax toxin receptors. A, B, Flow cytometry measuring protective antigen (PA) binding and internalization by human platelets (A) and by THP-1 cells (B) after 2 hours of treatment with 1000 ng/mL PA-biotin at 4°C, 37°C, or incubation in buffer alone. Cells were fixed in a 1:1 ratio of 3.7% formaldehyde, probed with fluorescein isothiocyanate (FITC)–streptavidin, and analyzed using FACScan (BD) and FCS Express (DeNovo) software. Data are representative of 4 experiments from 3 platelet donors and 3 experiments using separate THP-1 cell preparations. C, D, Flow cytometry measuring anthrax receptor surface expression in human platelets (C) and THP-1 cells (D). Cells were fixed, probed for tumor endothelial marker 8 (TEM8)/capillary morphogenesis gene 2 (CMG2), LRP6, or an isotype control, followed by secondary goat anti-rabbit Alexa Fluor 488–conjugated antibody, and analyzed using Accuri C6 (BD Biosciences) and FCS Express (De Novo). Data are representative of 3 experiments using 3 donors. E, Fold changes in peak fluorescent intensity of bound polyclonal antibodies directed against anthrax receptors: TEM8/CMG2 and LRP6 in human platelets and THP-1 cells. Brackets show the standard error of the mean for 3 donors. The fold change in peak fluorescence intensity for each condition was calculated by comparing each sample with the peak fluorescence intensity of an isotype control polyclonal primary rabbit antibody.

Article Snippet: Antibodies used were reactive against MEK1 (Upstate), TEM8/CMG2 (Abcam), low density lipoprotein receptor-related protein 6 (LRP6) (Santa Cruz), Alexa Fluor 488 goat anti-rabbit immunoglobulin G (H+L) (Invitrogen), isotype control/VASP antibody (Cell Signaling), total HSP27 (Cell Signaling), phosphorylated serine 82 HSP27 (Cell Signaling), or β-actin (Sigma).

Techniques: Binding Assay, Expressing, Flow Cytometry, Incubation, Software, Marker, Fluorescence