anti axl  (Cell Signaling Technology Inc)

Journal: Cancer Medicine

Article Title: TYRO3 , AXL , MERTK and Their Ligands in Brain Metastases From Colorectal Cancers

doi: 10.1002/cam4.71869

Figure Lengend Snippet: TAM receptors and ligands' protein expression profile in in vitro models of CRC and orthotopic in vivo model of BM‐CRC. (A) RNAseq data from cell lines presented per cancer type in nTPM (normalized number of transcripts per million) from the Human Protein Atlas database. (B) RNAseq data per colorectal cancer cell line in nTPM from the Human Protein Atlas. (C–G) RNAseq data for PROS1 (C), GAS6 (D), TYRO3 (E), AXL (F), and MERTK (G) in CL40, HT29, LS1034, SW1463, BM‐SC‐CRC1, and BM‐SC‐CRC2 cells. (H–N) Nude mouse brains injected with BM‐SC‐CRC1/2 cells, perfused with 4% PFA, and cut with a microtome (40 μm slices). The control condition (H) is in the absence of a primary antibody showing a unique lesion formed after injection. The red frames correspond to the fields of the higher magnifications (scale bar: Whole brain 2 mm, cropped image 50 μm). Immunostaining for TYRO3 (I), MERTK (J), PROS1 (K) and GAS6 (L) receptors and their ligands (×20 objective, Olympus VS120 slide scanner). The frontier between the human tumor cells and the mouse brain tissue is drawn by a dotted line (*: mouse brain; scale bar: 50 μm). Immunostaining for AXL without (M) and without (N) primary antibody. Scale bar: 50 μm. Nuclei were counterstained in blue.

Article Snippet: The primary antibodies used in our assays were directed against TYRO3 (rabbit polyclonal; #NBP2‐23725; Novus, Bio‐Techne; dilution 1:2000), AXL (goat polyclonal; #AF154; R&D Systems, Bio‐Techne; 1:400), MERTK (rabbit polyclonal; #MKT‐121AP; FabGennix; 1:800), PROS1 (rabbit polyclonal; #A0384; Dako; 1:800), and GAS6 (rabbit polyclonal; generous gift from Dr. Michael Hall [ ]; 1:2000).

Techniques: Expressing, In Vitro, In Vivo, RNA sequencing, Injection, Control, Immunostaining

Journal: Cancer Medicine

Article Title: TYRO3 , AXL , MERTK and Their Ligands in Brain Metastases From Colorectal Cancers

doi: 10.1002/cam4.71869

Figure Lengend Snippet: TAM receptors are expressed in a cohort of CRC patients with BM. Three micrometer paraffin slices of a tissue micro‐array from a cohort of 87 patients with CRC were immunostained for TYRO3, AXL, and MERTK ( n = 87, including n = 37 with BM; [ , , ]; blue Mayer's hematoxylin counterstaining). (A–C) Immunostaining scores were assigned from 1 to 4, according to the percentage of positive tumor cells, as shown in these representative images of TYRO3 (A), AXL (B), and MERTK (C) staining in biopsies from the tumor center. Arrows highlight blood vessels (scale bar: 300 μm; ×20 magnification on Olympus VS120 slide scanner). (D) Evolution of the immunostaining score from the primary tumor to the BM.

Article Snippet: The primary antibodies used in our assays were directed against TYRO3 (rabbit polyclonal; #NBP2‐23725; Novus, Bio‐Techne; dilution 1:2000), AXL (goat polyclonal; #AF154; R&D Systems, Bio‐Techne; 1:400), MERTK (rabbit polyclonal; #MKT‐121AP; FabGennix; 1:800), PROS1 (rabbit polyclonal; #A0384; Dako; 1:800), and GAS6 (rabbit polyclonal; generous gift from Dr. Michael Hall [ ]; 1:2000).

Techniques: Microarray, Immunostaining, Staining

Journal: Cancer Medicine

Article Title: TYRO3 , AXL , MERTK and Their Ligands in Brain Metastases From Colorectal Cancers

doi: 10.1002/cam4.71869

Figure Lengend Snippet: Prognostic value of TAM receptors in mCRC TCGA and BM‐CRC cohorts. Kaplan–Meier survival curves (A–C). TAM in the TCGA cohort of 85 patients with metastatic disease upon diagnosis. OS is defined as the time from primary tumor diagnosis. Patients were split according to the RNA expression level in the tumor (RSEM) into Low or High. Overall survival depending on the combined expression of TYRO3‐GAS6 (A), AXL‐GAS6 (B), MERTK‐GAS6 (C). (D–F) In the BM‐CRC cohort, patients were split according to the staining score in immunohistochemistry into Low (0, 1, 2) or High (3, 4). OS is the time from metastatic disease diagnosis; TYRO3 in the primary tumor (D), AXL in the primary tumor (E), MERTK in the primary tumor (F). (G–I) TYRO3 in BM (G), AXL in BM (H), MERTK in BM (I).

Article Snippet: The primary antibodies used in our assays were directed against TYRO3 (rabbit polyclonal; #NBP2‐23725; Novus, Bio‐Techne; dilution 1:2000), AXL (goat polyclonal; #AF154; R&D Systems, Bio‐Techne; 1:400), MERTK (rabbit polyclonal; #MKT‐121AP; FabGennix; 1:800), PROS1 (rabbit polyclonal; #A0384; Dako; 1:800), and GAS6 (rabbit polyclonal; generous gift from Dr. Michael Hall [ ]; 1:2000).

Techniques: Biomarker Discovery, RNA Expression, Expressing, Staining, Immunohistochemistry

Journal: Cancer Medicine

Article Title: TYRO3 , AXL , MERTK and Their Ligands in Brain Metastases From Colorectal Cancers

doi: 10.1002/cam4.71869

Figure Lengend Snippet: Graphical abstract. TAM receptors and their ligands in brain metastases from colorectal cancers. We assessed TAM receptors in different models. Created with Biorender.com . First, stem‐cell enriched BM‐derived cell lines from two CRC patients, CRC1 and CRC2. Orthotopic BM mice model and RNAseq data were generated, showing that AXL and its ligand GAS6 were poorly expressed in those stem‐cell enriched models. Second, we stained for TAM receptors on tissues from the primary tumor and metastatic sites in CRC1 and CRC2 patients, discovering that AXL was expressed in those tissues but on endothelial cells. TYRO3 was instead expressed on tumor cells and MERTK on leukocytes. Third, we stained for TAM receptors in a tissue micro‐array of primary tumor, non‐brain, and brain metastases from a local cohort of 85 patients. This confirmed the vascular location of AXL and showed the evolution of TAM protein expression from primary tumor to BM tissue. Last, we studied the prognostic properties of TAM receptors and their ligand in a TCGA cohort of patients with metastatic CRC. GAS6 appeared as a robust prognostic factor and suggested a prognostic impact of the balance between AXL and GAS6 expression as patients with tumors expressing Low AXL/High GAS6 had a significantly shortened overall survival. Finally, we provide a comprehensive study of TAM expression in metastatic CRC with a strong focus on BM‐CRC, underlining AXL expression evolution from primary tumor to BM and its prognostic potential in BM‐CRC. Although non‐significant, AXL proteic expression stratifies better the patients with BM‐CRC when assessed in brain tissue instead of primary tumor tissue. AS compared with TYRO3 and MERTK, AXL proteic expression is more frequently conserved, meaning increased or stable, from primary tumor to brain metastasis.

Article Snippet: The primary antibodies used in our assays were directed against TYRO3 (rabbit polyclonal; #NBP2‐23725; Novus, Bio‐Techne; dilution 1:2000), AXL (goat polyclonal; #AF154; R&D Systems, Bio‐Techne; 1:400), MERTK (rabbit polyclonal; #MKT‐121AP; FabGennix; 1:800), PROS1 (rabbit polyclonal; #A0384; Dako; 1:800), and GAS6 (rabbit polyclonal; generous gift from Dr. Michael Hall [ ]; 1:2000).

Techniques: Derivative Assay, RNA sequencing, Generated, Staining, Microarray, Expressing