recombinant mouse timp 1 protein r d systems  (R&D Systems)

Journal: Cancer Discovery

Article Title: TIMP1 Mediates Astrocyte-Dependent Local Immunosuppression in Brain Metastasis Acting on Infiltrating CD8 + T Cells

doi: 10.1158/2159-8290.CD-24-0134

Figure Lengend Snippet: TIMP1 mediates brain metastasis in a CD8 + T-cell–dependent manner. A, Schema of the experimental design. pSTAT3 − and pSTAT3 + wt and pSTAT3 + cKO GFAP - Timp1 CM (with or without rTIMP1 100 ng/mL or control IgG/anti-TIMP1 10 μg/mL) was added to CD8 + T cells and cultured with BrM cells in a 1:4 ratio (BrM-OVA cancer cells: OT-I T cells specific for the OVA-derived SIINFEKL peptide) or a 1:5 ratio (BrM cancer cells:CD8 + T cells previously activated). B, Quantification of the BLI signal from the experiment shown in A and representative images of B16/F10-BrM-OVA–derived BLI at the initial time point and 24 hours after adding CD8 + lymphocytes preincubated with CM. Light orange condition refers to coculture of OT-I T cells with B16/F10-BrM no OVA (control for antigen-specific killing). Values correspond to 24 hours BLI normalized to BLI before adding CD8 + T cells expressed in percentage with respect to the mean of control experimental condition (BrM cells). Error bars, SEM. n = 3 different cocultures per condition. P value was calculated using the two-tailed t test. C and D, Schema of the experimental design. Control IgG or anti-TIMP1 (10 μg/mL) was added to the medium in organotypic cultures of mouse brain with B16/F10-BrM established lesions ( C ) and PDOC that include the brain metastasis–associated microenvironment ( D ). E, Quantification of the BLI signal emitted by B16/F10-BrM cells in each brain slice normalized by the initial value obtained at day 0, before the addition of control IgG, anti-TIMP1 (10 μg/mL) or anti-CD8 (100 μg/mL). Values are shown in box-and-whisker plots in which every dot represents a different organotypic culture and the line in the box corresponds to the median. Whiskers go from minimum to maximum values ( n = 42 IgG, 39 anti-TIMP1 and 27 anti-TIMP1 plus anti-CD8 independent organotypic cultures). Quantification is accompanied by representative images of wells containing brain organotypic cultures with established B16/F10-BrM metastases grown ex vivo for 3 days. The image shows the BLI intensity in each condition for each brain slice. P values were calculated using the two-tailed t test. F, Quantification of the number of Ki67 + cancer cells found in IgG2 and anti-TIMP1-treated PDOCs. Values are shown in box-and-whisker plots in which every dot represents a patient and each patient is an independent experiment ( n = 11). The pie chart shows all BrM-PDOCs quantified in the graph and classified according to the specific primary tumor. P value was calculated using two-tailed t test. G, Quantification of the number of Ki67 + cancer cells found in IgG2, anti-TIMP1 (10 μg/mL), and anti-TIMP1 (10 μg/mL) plus anti-CD8 (10 μg/mL) PDOCs. Values are shown in box-and-whisker plots in which every dot represents a patient and each patient is an independent experiment ( n = 7). P value was calculated using two-tailed t test. H, Schema of the experimental design. cKO GFAP - Timp1 mice were inoculated with BrM cells intracardially, and after 2 weeks, ex vivo brain BLI was analyzed. I and J, Representative images of brains from control and cKO GFAP - Timp1 mice intracardially injected with B16/F10-BrM ( I ) or E0771-BrM ( J ) cells. The image shows the BLI intensity in each condition. K and L, Quantification of ex vivo brain BLI. Values are shown in box-and-whisker plots in which every dot represents a different animal. Values were obtained from normalizing the ex vivo brain signal to the in vivo head signal 3 days after intracardiac injection with either B16/F10-BrM ( K ) or E0771-BrM ( L ) cells ( n = 26/29 mice four independent experiments in K and n = 28/25 mice three independent experiments in L ). P value was calculated using the two-tailed t test. M, Representative images of CD8 + T cells in metastatic lesions growing in the brains from control or cKO GFAP - Timp1 mice intracardially injected with E0771-BrM at experimental endpoint. White arrowhead indicates CD8 + T cells and red arrowheads indicate Ki67 + CD8 + T cells. Scale bar, 25 μm, magnification 5 μm. N, Quantification of the total number of CD8 + T cells in control and cKO GFAP - Timp1 mice intracardially injected with E0771-BrM at human endpoint. Values are shown in box-and-whisker plots in which every dot represents a different animal. Ten brains were analyzed in each condition. P value was calculated using the two-tailed t test.

Article Snippet: Anti-TIMP1 antibody (102D1; 10 μg/mL, Thermo Fisher Scientific, cat.# MS608PABX) or rTIMP1 (100 ng/mL, R&D Systems, cat.# 980-MT) was added at day 0 when indicated.

Techniques: Control, Cell Culture, Derivative Assay, Two Tailed Test, Slice Preparation, Whisker Assay, Ex Vivo, Injection, In Vivo