Journal: NPJ Vaccines

Article Title: The co-delivery of adenovirus-based immune checkpoint vaccine elicits a potent anti-tumor effect in renal carcinoma

doi: 10.1038/s41541-023-00706-x

Figure Lengend Snippet: a , b The proliferation of lymphocytes isolated from the spleens of mice in each group after continuous stimulation of CAIX protein (10 μg/ml) was detected by the EdU assay. c Detection of T lymphocytes secreting IFN-γ by ELISPOT assay. d After stimulation with CAIX protein, the proportions of IL-2 + CD8 + T cells, IFN-γ + CD8 + T cells, and TNF-α + CD8 + T cells in splenocytes from each group were detected by flow cytometry. e – g Statistical analysis of the percentages of IL-2 + CD8 + T cells, TNF-α + CD8 + T cells, and IFN-γ + CD8 + T cells in d . Data presented as mean ± SD from one representative experiment of three performed. The different significance was set at *** p < 0.001 and **** p < 0.0001. Multiple groups of comparison data were analyzed by one-way ANOVA.

Article Snippet: For cell surface staining, cells were incubated with the following antibodies: PE-conjugated anti-human CAIX (R&D Systems, Cat. FAB2188P, 1:100), PE-conjugated anti-mouse CD3ε (BioLegend, Cat. 100308, 1:100), APC-conjugated anti-mouse PD-L1 (BioLegend, Cat. 124312, 1:100), APC-conjugated anti-mouse CD11c (BioLegend, Cat. 117310, 1:100), PerCP-Cy5.5-conjugated anti-mouse CD4 (BioLegend, Cat. 116012, 1:100), PerCP-Cy5.5-conjugated anti-mouse CD8α (BioLegend, Cat. 100734, 1:100), FITC anti-mouse CD49b (BioLegend, Cat. 108906, 1:100), PerCP-conjugated anti-mouse F4/80 (BioLegend, Cat. 123126, 1:100), FITC-conjugated anti-mouse CD11b (BioLegend, Cat. 101206, 1:100) for 1 h at 4 °C.

Techniques: Isolation, EdU Assay, Enzyme-linked Immunospot, Flow Cytometry, Comparison

Journal: NPJ Vaccines

Article Title: The co-delivery of adenovirus-based immune checkpoint vaccine elicits a potent anti-tumor effect in renal carcinoma

doi: 10.1038/s41541-023-00706-x

Figure Lengend Snippet: Ad-CAIX and Ad- PD-L1 vaccines were prepared and expressed in vitro. Three tumor models, including the subcutaneous, lung metastasis, and orthotropic tumor, were established, and intramuscular Ad vaccine immunization was performed. Ad-CAIX/Ad-PD-L1 could effectively enhance the induction and maturation of DCs and DC subsets, and promote strong tumor-specific CD8 + T cell immune responses. Ad-CAIX/Ad-PD-L1 vaccine could significantly inhibit tumor growth or lung metastasis in three models via DCs-mediated CD8 + T cell anti-tumor responses.

Article Snippet: For cell surface staining, cells were incubated with the following antibodies: PE-conjugated anti-human CAIX (R&D Systems, Cat. FAB2188P, 1:100), PE-conjugated anti-mouse CD3ε (BioLegend, Cat. 100308, 1:100), APC-conjugated anti-mouse PD-L1 (BioLegend, Cat. 124312, 1:100), APC-conjugated anti-mouse CD11c (BioLegend, Cat. 117310, 1:100), PerCP-Cy5.5-conjugated anti-mouse CD4 (BioLegend, Cat. 116012, 1:100), PerCP-Cy5.5-conjugated anti-mouse CD8α (BioLegend, Cat. 100734, 1:100), FITC anti-mouse CD49b (BioLegend, Cat. 108906, 1:100), PerCP-conjugated anti-mouse F4/80 (BioLegend, Cat. 123126, 1:100), FITC-conjugated anti-mouse CD11b (BioLegend, Cat. 101206, 1:100) for 1 h at 4 °C.

Techniques: Vaccines, In Vitro

Journal: Journal of Clinical Investigation

Article Title: Tumor endothelial marker 1–specific DNA vaccination targets tumor vasculature

doi: 10.1172/jci67382

Figure Lengend Snippet: Figure 4 Tem1-TT vaccination inhibits CT26 tumor vascularization. (A) Tem1-TT vaccination reduced tumor hemoglobin content. Tumors at approximately 200 mm3 were excised from TT- or Tem1-TT–vaccinated mice and inspected grossly. Tumors from Tem1-TT–vaccinated mice appeared pale relative to control tumors. Reduced hemoglobin levels in tumors from Tem1-TT–vaccinated mice were observed by ELISA. (B) Tem1-TT vaccine reduces tumor vascularity. Tumors from TT- or Tem1-TT–vaccinated mice were analyzed by Doppler ultrasound. Perfused tumor area and real blood flux are shown, measured and calculated by Doppler image analysis. (C) CT26 tumors from Tem1-TT–immunized mice had significantly decreased CD31 expression compared with TT vaccination. Also note the abnormal blood vessel shape. Original magnification, ×20. (D) CAIX expression was increased in tumors from Tem1-TT–immunized animals. CAIX expression was visualized by immunohistochemistry, and Caix was independently quantified by qRT-PCR in tumors from mice vaccinated with either TT or Tem1-TT. Original magnification, ×20. Data in A–D are mean ± SD of a representative experiment (n = 5 per group). Statistical analyses were performed with Student’s t test.

Article Snippet: Sections (6 μm thick) were stained for mouse CAIX (R&D Systems; catalog no. AF2344, DAB chromogen), CD31 (BD Biosciences — Pharmingen; clone 390, catalog no. 558737, DAB chromogen), and CD3 (BD Biosciences — Pharmingen; clone 15.5-2C11, catalog no. 550275, DAB chromogen) with hematoxylin as a counterstain.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry, Quantitative RT-PCR

Journal: Cancers

Article Title: Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts

doi: 10.3390/cancers11071002

Figure Lengend Snippet: Sunitinib induces hypoxia and Carbonic Anhydrase IX (CAIX) expression in primary Triple Negative Breast Cancer (TNBC) tumors. ( a ) Images of MDA-MB-231 LM2-4Luc + primary tumor tissue sections harvested at increasing tumor volumes from animals administered either vehicle or 60 mg/kg sunitinib and immunohistochemically stained for the indicated markers. Scale bars: upper panels, 1 mm; lower panels, 200 μm. ( b to e ) Image-based quantification of ( b ) CD31 + blood vessels, ( c ) CAIX expression, ( d ) pimonidazole and ( e ) proliferation. Data show the mean ± standard error of the mean (SEM). n = 3 animals/group with 10 images/animal. * p < 0.05, *** p < 0.001.

Article Snippet: Formalin-fixed, paraffin-embedded (FFPE) tissues were sectioned (4–5 μm) and stained with primary antibodies against human CAIX (1:50, AF2188; R&D Systems, Minneapolis, MN, USA), CD31 (1:10, DIA-310; Histobiotec, Miami Beach, FL, USA), BrdU (1:10, Roche, Laval, QC, Canada) and pimonidazole (1:100, Hydroxyprobe, Burlington, MA, USA).

Techniques: Expressing, Staining

Journal: Cancers

Article Title: Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts

doi: 10.3390/cancers11071002

Figure Lengend Snippet: SLC-0111 reduces vessel density and vascular permeability in primary tumors, and decreases lung and liver metastases. ( a ) Representative 3D maximum projections of whole mount primary tumor tissue slices showing CD31 + blood vessels (green) and fluorescently labeled dextran (red). Merged images demonstrate the presence of both intravascular (yellow) and extravasated (arrowheads) dextran. Scale bar = 100 μm. ( b ) Quantification of the number of vessels in whole mount primary tumor slices. Data show the mean ± standard error of the mean (SEM). n = 13–14 images/group. ** p < 0.01, *** p < 0.001. ( c ) Quantification of vascular permeability as assessed by relative area of extravasated dextran in whole mount primary tumor slices. Data show the mean ± SEM. n = 13–16 images/group. * p < 0.05, ** p < 0.01. ( d ) Representative 3D maximum projections of whole mount primary tumor tissue slices showing levels of CAIX expression (green) and the number of CD31 + blood vessels (red). Lower panels, merge. Scale bar = 100 μm. ( e ) Quantification of Carbonic Anhydrase IX (CAIX) expression in whole mount primary tumor tissue slices in panel d. Data show the mean ± SEM. n = 11–15 images/group. * p < 0.05, *** p < 0.001. ( f ) Representative images of lung and liver tissues from animals with MDA-MB-231 LM2-4Luc + orthotopic breast tumors and administered SLC-0111 and sunitinib, either alone or in combination. Visible metastatic nodules (arrows) are indicated. Scale bar = 1 cm. ( g – i ) Analysis of ( g ) lung weight ( n = 9/group), ( h ) liver weight as a function of whole animal body weight ( n = 9/group) and ( i ) number of metastatic nodules present on the liver surface ( n = 7–8/group). For each graph, data show the mean ± SEM. * p < 0.05.

Article Snippet: Formalin-fixed, paraffin-embedded (FFPE) tissues were sectioned (4–5 μm) and stained with primary antibodies against human CAIX (1:50, AF2188; R&D Systems, Minneapolis, MN, USA), CD31 (1:10, DIA-310; Histobiotec, Miami Beach, FL, USA), BrdU (1:10, Roche, Laval, QC, Canada) and pimonidazole (1:100, Hydroxyprobe, Burlington, MA, USA).

Techniques: Permeability, Labeling, Expressing

Journal: Cancers

Article Title: Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts

doi: 10.3390/cancers11071002

Figure Lengend Snippet: Administration of SLC-0111 reduces lung metastases. ( a ) Representative 3D maximum projections of whole mount lung tissue slices from tumor-bearing mice, showing vimentin-positive metastases (cyan) and CD31 + blood vessels (red). Scale bar = 100 μm. ( b ) Analysis of vimentin positive metastases in whole mount lung tissue sections described in panel a. Data show the mean ± standard error of the mean (SEM). n = 8–10 images/group. * p < 0.05, *** p < 0.001. ( c ) Representative 3D maximum projections of whole mount lung tissue slices from tumor-bearing mice, showing Carbonic Anhydrase IX (CAIX)-positive metastases (green) and CD31 + blood vessels (red). Bar = 100 μm. ( d ) Analysis of CAIX positive metastases in the whole mount lung tissue sections described in panel c. Data show the mean ± SEM. n = 5–8 images/group: No statistically significant differences were observed among the groups.

Article Snippet: Formalin-fixed, paraffin-embedded (FFPE) tissues were sectioned (4–5 μm) and stained with primary antibodies against human CAIX (1:50, AF2188; R&D Systems, Minneapolis, MN, USA), CD31 (1:10, DIA-310; Histobiotec, Miami Beach, FL, USA), BrdU (1:10, Roche, Laval, QC, Canada) and pimonidazole (1:100, Hydroxyprobe, Burlington, MA, USA).

Techniques:

Journal: Cancers

Article Title: Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts

doi: 10.3390/cancers11071002

Figure Lengend Snippet: Model for targeting angiogenesis and Carbonic Anhydrase IX (CAIX) to reduce metastasis of Triple Negative Breast Cancer (TNBC). Exposure of tumors to anti-angiogenic agents such as sunitinib leads to reduced tumor growth. However, major consequences can include enhanced hypoxia and increased vascular permeability. The exacerbation of hypoxia results in the upregulation of CAIX expression by tumor cells and the potentiation of metastasis. Inhibition of CAIX activity inhibits metastasis through several mechanisms, as shown by previous studies, but may also play a role in reducing vascular permeability and contributing to vascular normalization, potentially reducing the invasion of tumor cells to distant sites.

Article Snippet: Formalin-fixed, paraffin-embedded (FFPE) tissues were sectioned (4–5 μm) and stained with primary antibodies against human CAIX (1:50, AF2188; R&D Systems, Minneapolis, MN, USA), CD31 (1:10, DIA-310; Histobiotec, Miami Beach, FL, USA), BrdU (1:10, Roche, Laval, QC, Canada) and pimonidazole (1:100, Hydroxyprobe, Burlington, MA, USA).

Techniques: Permeability, Expressing, Inhibition, Activity Assay

Journal: Journal of Translational Medicine

Article Title: Are morphological criteria sufficient for the identification of circulating tumor cells in renal cancer?

doi: 10.1186/1479-5876-11-214

Figure Lengend Snippet: Summary of the immunocytochemical analysis of CNHCs with antibodies against CD45, CD31, and CAIX

Article Snippet: The filters were incubated with primary antibodies directed against CAIX (rabbit IgG, NB100-417, Novus Biologicals, Littleton, USA; 3.5 μg/ml ), CD31 (mouse IgG1, M0823, Dako, Glostrup, Denmark; 1 μg/ml) or CD45 (mouse IgG1, M0855, Dako, Glostrup, Denmark; 2.4 μg/ml) diluted in Antibody Diluent (Dako, Glostrup, Denmark) for 30 min at RT followed by application of primary antibody enhancer (Dako, Glostrup, Denmark) for 15 min.

Techniques:

Journal: Journal of Translational Medicine

Article Title: Are morphological criteria sufficient for the identification of circulating tumor cells in renal cancer?

doi: 10.1186/1479-5876-11-214

Figure Lengend Snippet: Immunocytochemical analysis of CNHCs with antibodies against the RCC marker CAIX. Clusters of CNHCs cytomorphologically classified as uncertain malignant (−UMF) with cytoplasmic positive staining with antibodies against the RCC marker CAIX (A) . Clusters of CNHC-UMF and -BF without reactivity for CAIX antibodies ( B and C , respectively). A single CNHC-MF with positive cytoplasmic (D) and without staining for CAIX (E) . Single CAIX-negative CNHC-UMF and -BF ( F and G , respectively).

Article Snippet: The filters were incubated with primary antibodies directed against CAIX (rabbit IgG, NB100-417, Novus Biologicals, Littleton, USA; 3.5 μg/ml ), CD31 (mouse IgG1, M0823, Dako, Glostrup, Denmark; 1 μg/ml) or CD45 (mouse IgG1, M0855, Dako, Glostrup, Denmark; 2.4 μg/ml) diluted in Antibody Diluent (Dako, Glostrup, Denmark) for 30 min at RT followed by application of primary antibody enhancer (Dako, Glostrup, Denmark) for 15 min.

Techniques: Marker, Staining

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: Identification of the CAIX Interactome using the BioID proximal ligation strategy and validation of high confidence proximal CAIX-associating proteins by co-immunoprecipitation. ( a ) Schematic showing the domain structure of the CAIX-BirA* fusion protein used for the BioID studies. SP, signal peptide; PG, proteoglycan-like domain; CA, catalytic domain; TM, transmembrane domain; IC, intracellular domain. ( b ) FACS analysis showing levels of constitutive expression of the CAIX-BirA* fusion protein expressed by MDA-MB-231 cells (MDA-MB-231-CAIX-BirA*) cultured in normoxia compared to levels of endogenous CAIX expression by WT MDA-MB-231 cells cultured in either normoxia or hypoxia. The filled (gray) histograms indicate specific CAIX staining whereas the dotted lined histograms indicate staining by isotype-specific control IgG. ( c ) Image showing exogenous CAIX (red) localized at the cell surface of MDA-MB-231-CAIX-BirA* cells grown in normoxia. Scale bar, 10 μ M . ( d – g ) Co-IP of high confidence proximal CAIX-associating proteins ( d ) ITGB1, ( e ) ITGA2 ( f ) CD98hc and ( g ) MMP14 with exogenous CAIX expressed by MDA-MB-231-CAIX-BirA* cells cultured in normoxia. ( h – j ) Co-IP of ( h ) ITGB1, ( i ) ITGA2 and ( j ) MMP14 with endogenous CAIX expressed by WT MDA-MB-231 cells cultured in either normoxia (N) or hypoxia (H). Isotype-specific IgG was used as a control for co-IPs and data in ( d ) to ( j ) are representative of at least two independent experiments. IgG, immunoglobulin G.

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Ligation, Biomarker Discovery, Immunoprecipitation, Expressing, Cell Culture, Staining, Control, Co-Immunoprecipitation Assay

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: Selected CAIX-BirA*FLAG high-confidence proximal associating proteins identified by BioID in MDA-MB-231 cells

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques:

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX colocalizes with integrins and MMP14 in pseudopodia-like protrusions resembling lamellipodia. ( a ) Images of MDA-MB-231-CAIX-BirA* cells cultured on type 1 collagen in normoxia, showing colocalization (yellow, arrows in merge) of exogenously expressed CAIX (red) with ITGB1, ITGA2, MMP14 and cofilin (each marker in green), but not with FAK (green; arrowheads). Scale bar, 10 μm. ( b ) Images of WT MDA-MB-231 cells grown in either normoxia or hypoxia, showing colocalization of hypoxia-induced CAIX (red) with F-actin (green) in pseudopodia-like protrusions (arrows, merge). Scale bar, 10 μm. ( c ) Images of WT MDA-MB-231 cells cultured on type 1 collagen in hypoxia, showing colocalization of hypoxia-induced CAIX (red) with F-actin, ITGA2 and MMP14 (green) at leading edges of cells (arrows, merge), but not at focal adhesions. Scale bar, 10 μm. ( d and e ) 3D maximum projections of 2D images of MDA-MB-231-CAIX-BirA* cells transfected with GFP and cultured on type 1 collagen in normoxia, showing robust colocalization of CAIX (red) with ( d ) ITGA2 and ( e ) MMP14 (blue) in pseudopodia-like protrusions and depletion of cytoplasmic GFP (green) from these regions. Images for each channel are shown in the upper panels and merged images are shown in the lower panels. Scale bar, 10 μm.

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Cell Culture, Marker, Transfection

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX regulates migration of breast cancer cells. ( a ) Images of wound-induced cell migration by the indicated MDA-MB-231 cells cultured in normoxia. Scale bar, 300 μm. Dashed lines demarcate the wound boundary at t =0 h and yellow shading indicates the cell-free area. ( b ) Quantification of migration by the cells described in a . *** P <0.001. ( c ) Images of wound-induced migration of MDA-MB-231 CAIX-BirA* cells cultured in normoxia and treated with increasing concentrations of U-104. Scale bar, 300 μm. Wound boundaries and cell-free area are demarcated as described in a . ( d ) Quantification of migration by the cells described in c . * P <0.05, *** P <0.001. Data in panels ( b ) and ( d ) show the mean±s.e.m. of technical replicates ( n =16) and are representative of two independent experiments. ( e ) Wound-induced migration of WT MDA-MB-231 cells cultured in normoxia (black bars) or hypoxia (gray bars) and treated with U-104. Data show the mean±s.e.m. of technical replicates ( n =12) and are representative of three independent experiments. ** P <0.01, *** P <0.001. Statistical analysis was performed using Student’s t -test ( b ) or ANOVA ( d , e ).

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Migration, Cell Culture

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX colocalizes with MMP14 at invadopodia and regulates type I collagen degradation. ( a ) Images of a MDA-MB-231-CAIX-BirA* cell (white dashed line shows boundary) cultured on type I collagen in normoxia, identifying a ROI (yellow box) containing invadopodia (arrows) labeled for Cortactin (cyan), CAIX (red) and MMP14 (green). Profile plots showing colocalization of CAIX and MMP14 at cortactin-positive invadopodia (1 and 2) along the indicated lines (insets) are shown at the right. Scale bar, 10 μm. ( b ) Images of an MDA-MB-231-CAIX-BirA* cell (white dashed line shows cell boundary) cultured on DQ type I collagen in normoxia, identifying an ROI (yellow box) containing regions of collagen degradation (1 and 2, arrows) that are labeled for DQ collagen (cyan), CAIX (red) and MMP14 (green). Profile plots showing colocalization of the signals along the indicated lines (insets) are shown to the right. Scale bar, 10 μm. ( c ) Images showing WT MDA-MB-231 cells cultured in hypoxia on fluorescently labeled gelatin (blue, with black areas denoting regions of degradation) and stained for CAIX (green) and markers of invadopodia (red), including cortactin, Tks5 and MMP14 to demonstrate colocalization at mature invadopodia (white arrows; yellow foci). Scale bars, 10 μm (left), 2 μm (right). ( d ) Quantification of DQ collagen degradation by the indicated MDA-MB-231 cell lines grown in normoxia. *** P <0.001. ( e ) Quantitation of degradation of DQ collagen by MDA-MB-231 CAIX KO and CAIX rescue cells cultured in normoxia. * P <0.05. Data in ( d ) and ( e ) show the mean±s.e.m. of technical replicates ( n =5) and are representative of three independent experiments. ( f ) Dose-dependent inhibition of DQ collagen degradation by CAIX rescue cells treated with U-104. Data show the mean±s.e.m. of technical replicates ( n =4) and are representative of two independent experiments. * P <0.05, ** P <0.01. ( g ) Quantification of DQ collagen degradation by WT MDA-MB-231 and CAIX KO cells cultured in hypoxia. Data show the mean±s.e.m. of technical replicates ( n =8) and are representative of three independent experiments. ** P <0.01. ( h and i ) Invasion through Matrigel by highly metastatic MDA-MB-231 LM2-4 breast cancer cells cultured in hypoxia and ( h ) expressing CAIX-targeted (shCAIX) shRNA or ( i ) treated with U-104. Data show the mean±s.e.m. of three independent experiments. *** P <0.001. Western blots of hypoxia-induced CAIX expression are shown as insets.

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Cell Culture, Labeling, Staining, Quantitation Assay, Inhibition, Expressing, shRNA, Western Blot

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX regulates breast cancer cell invasion and metastasis. ( a ) Schematic showing the domain structure of wild type (WT) huCAIX (459 aa) and constructs lacking either the intracellular domain (ΔIC), the extracellular proteoglycan-like domain (ΔPG) or the indicated point mutations in the IC domain. SP, signal peptide; CA, catalytic domain; TM, transmembrane domain. ( b ) Western blot analysis of the levels of expression of the indicated huCAIX constructs by 4T1shCAIX cells cultured in hypoxia. β-actin served as a loading control. ( c ) Analysis of CAIX catalytic activity using the in-cell carbonic anhydrase activity assay. Assays were performed in normoxia in the presence or absence of U-104 (50 μ M ) as indicated. Levels of CAIX catalytic activity were normalized by using the time required to achieve 50% of the total decrease in pH. Data were normalized to the spontaneous rate of reaction in the presence of buffer alone and the activity of cells expressing WT huCAIX was set to 100%. Data show the mean±s.e.m. of technical replicates ( n =3/group) and are representative of three independent experiments * P <0.05, *** P <0.001. ( d ) Invasion through Matrigel by the indicated 4T1 cell lines cultured in hypoxia. Data show the mean±s.e.m. of three independent experiments. * P <0.05, *** P <0.001. ( e ) Invasion through Matrigel by the 4T1shCAIX cell lines expressing WT, ΔIC and ΔPG variants of huCAIX and cultured in hypoxia. * P <0.05, *** P <0.001. ( f ) Invasion through Matrigel by 4T1 sh/WT huCAIX cells cultured in hypoxia and treated with U-104. *** P <0.001. Western blots of hypoxia-induced CAIX expression are shown as insets. For ( d )– ( f ), data show the mean±s.e.m. of three independent experiments. ( g – i ) Analysis of invasion through type 1 collagen by 4T1 sh/WT huCAIX cells cultured in hypoxia and treated with ( g ) U-104 (50 μ M ), ( h ) anti-MMP14 antibody (20 μg/ml) or ( i ) a combination of anti-MMP14 antibody (20 μg/ml) and U-104 (50 μ M ). * P <0.05, ** P <0.01. Data in ( g )–( i ) show the mean±s.e.m. of three independent experiments. ( j ) Analysis of spontaneous lung metastases formed by the indicated 4T1 cell lines following growth of orthotopic breast tumors. Data show the mean±s.e.m. n =6/group. ( k ) Analysis of experimental lung metastases formed by the indicated 4T1 cell lines. Mean±s.e.m. is shown. n =6/group. * P <0.05, ** P <0.01. Statistical analysis was performed using ANOVA ( c , d , e , k ) or Student’s t -test ( f , g , h ).

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Construct, Western Blot, Expressing, Cell Culture, Control, Activity Assay

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX associates with MMP14 through its intracellular domain and potentiates MMP14 activity. ( a ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT, ΔIC or ΔPG variants of huCAIX. ( b ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT huCAIX or the T443A point mutant. ( c ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT or the Y449A point mutant. ( d ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells grown in hypoxia and expressing WT huCAIX, the S448A point mutant or the ΔPG mutant. ( e ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells in hypoxia expressing WT huCAIX, the ΔIC truncation or the double point mutant S448A+Y449A. Isotype-specific IgG was used as a control for co-IPs. ( f ) Time-dependent stimulation of MMP14 activity in the presence (red bars) or absence (black bars) of equimolar concentrations of CAIX. Data show the mean±s.e.m. of technical replicates ( n =5) and are representative of two independent experiments. * P <0.05, ** P <0.01. ( g ) Dose-dependent inhibition of CAIX-mediated stimulation of MMP14 activity in the presence of increasing concentrations of U-104. Mean±s.e.m. of technical replicates ( n =5). Data are representative of two independent experiments. *** P <0.001. ( h ) Inhibition of CAIX-mediated stimulation of MMP14 activity in the presence of increasing amounts of D 2 O (heavy water). Mean±s.e.m. of technical replicates ( n =5). Data are representative of three independent experiments. *** P <0.001. Statistical analysis was performed using Student’s t -test ( f ) or ANOVA ( g , h ).

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Cell Culture, Expressing, Mutagenesis, Control, Inhibition

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX stimulates the activity of MMP14. Model for CAIX-mediated regulation of MMP14 activity. CAIX, which is upregulated in hypoxia, associates through its intracellular domain with MMP14 at functional invadopodia. At these invasive structures, CAIX functions to potentiate MMP14-mediated collagen degradation by directly contributing H + ions required for MMP14 catalytic activity.

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Activity Assay, Functional Assay

Journal: bioRxiv

Article Title: Cell Programmed Nutrient Partitioning in the Tumor Microenvironment

doi: 10.1101/2020.08.10.238428

Figure Lengend Snippet: a-b , Representative OCR ( a ) and ECAR ( b ) tracings of MC38 tumor cell fractions with indicated injections of oligomycin (O), FCCP (F), and rotenone and antimycin A (R/AA). c-d Basal mitochondrial OCR ( c ) and cellular ECAR ( d ) of MC38 tumor fractions (n=5 mice). e , Unsupervised cluster analysis of differentially expressed metabolic mRNA transcripts of whole tumor, CD45-cancer cell, CD45+ CD11b+ F4/80hi TAM, CD45+ CD11b+ F4/80lo myeloid cell, CD45+ CD3+ CD8a+ T cell, and CD45+ CD3+ CD8a-(CD4+) T cell flow-sorted MC38 tumor populations (n=2 mice). f-g , Glucose transporter ( f ) and hexokinase ( g ) mRNA transcript levels of indicated MC38 tumor populations (n=2 mice). Dotted line approximates limit of detection. hi , Representative flow cytometry plots ( h ) and quantification ( i ) of pS6 levels in indicated MC38 tumor and spleen populations. j-k , Representative flow cytometry histograms ( j ) and quantification ( k ) of pS6 levels in cancer cells (CD45-CA9+), myeloid cells (CD45+ CD11b+ CD14+), T cells (CD45+ CD3+), and other immune cells (CD45+ CD3-CD14-) from patient ccRCC tumor and PBMC (n=4 patients). Each data point represents a biological replicate and error bars are SEM. a-d and f-g are representative of at least two independent experiments. P values were calculated using Welch’s 2-tailed t-test for (c-d) and Brown-Forsythe ANOVA test for (h, j). * p <0.05, ** p <0.01, *** p <0.001. ECAR: extracellular acidification rate; FCCP: Trifluoromethoxy carbonylcyanide phenylhydrazone; FMO: fluorescence minus one; MFI: median fluorescence intensity; OCR: oxygen consumption rate; PBMC: peripheral blood mononuclear cells; pS6: phosphorylated ribosomal protein S6 (Ser235/236).

Article Snippet: The antihuman antibodies used were: CD45 BV421 (HI30, BioLegend 304032), CD3 APC (UCHT1, BioLegend 300439), CD11b PerCP-Cy5.5 (ICRF44, BioLegend 301328), CD14 BV510 (M5E2, BioLegend 301842), CA9 AF647 (303123, R&D Systems FAB2188R-100UG), Human Fc Block (BD 564220).

Techniques: Flow Cytometry, Fluorescence

Journal: Redox Biology

Article Title: Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia

doi: 10.1016/j.redox.2019.101297

Figure Lengend Snippet: CA9 overexpression in MM cells correlates with increased catalytic Fe(II) in response to hypoxia . (A) Differential overexpression of Ca(r)9 in asbestos-induced rat MM previously analyzed by expression array. Normal mesothelium (N = 2)/Met-5A cells (N = 2) in combination (N = 4) were used as a control; SM (N = 13) or EM (N = 9) represent sarcomatoid or epithelioid subtypes of malignant mesothelioma (MM) [ , ]. (B) Ca(r)9 overexpression in MM samples (shown in A plus biphasic subtype; N = 2; total N = 24) exhibited an inverse correlation with rat survival . Pearson's correlation coefficient and corresponding P value is indicated. Quantitative RT-PCR (C) using GAPDH and western blotting (D) using β-actin, as corresponding internal standards, were performed to access CA9 mRNA and protein levels by using two human EM (ACC-Meso-1 and NCI–H2052) and one SM (NCI–H2373) cell lines in comparison to non-tumorous human mesothelial cell (MeT-5A) after 48 h-incubation in normoxia (N) or hypoxia (H; 1% O 2 ). (E) Western blotting was performed to determine levels of iron metabolism-associated proteins in MM cells after 48 h-incubation in normoxia or hypoxia. (F) Differential levels of catalytic Fe(II) in normoxia or hypoxia of ACC-Meso-1 cells were determined by staining with SiRhoNox-1 (5 μM), followed by evaluation with a flow cytometer after 48 h-incubation. (G) Localization of catalytic Fe(II) in normoxic or hypoxic ACC-Meso-1 cells were analyzed by co-staining of SiRhoNox-1 (5 μM), LysoTracker (200 μM) and MitoTracker (75 μM) after 48 h-incubation and observation with a confocal microscope. Integrated intensity per cell (N = 7 ) was quantified by ZEN Imaging Software (ZEISS) (means ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 vs each control unless indicated by bar). Refer to text for details. SM, sarcomatoid mesothelioma; EM, epithelioid mesothelioma; N, normoxia; H, hypoxia; A.U., arbitrary unit.

Article Snippet: Mouse monoclonal anti-CA9 antibody (NBP1-51691) was from Novus Biologicals (Centennial, CO).

Techniques: Over Expression, Expressing, Control, Quantitative RT-PCR, Western Blot, Comparison, Incubation, Staining, Flow Cytometry, Microscopy, Imaging, Software

Journal: Redox Biology

Article Title: Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia

doi: 10.1016/j.redox.2019.101297

Figure Lengend Snippet: CA9 inhibition antagonizes MM cell growth and migration under hypoxia. ACC-Meso-1 and MeT-5A cells were cultured under hypoxia and treated with two CA9 inhibitors (S4 and U104). MTT (A) or WST-1 (B) assay was performed to evaluate anti-proliferation effect resulted from CA9 inhibitors in ACC-Meso-1 cells. MeT-5A was used as a non-tumorous control to evaluate the cytotoxic effect caused by CA9 inhibitors. (C) Wound healing assay was performed to estimate migration ability of ACC-Meso-1 cells in response to CA9 inhibition. Cell migration was evaluated by measurement of wound closure prior to (0 h) and after incubation with CA9 inhibitors (24 h). DMSO (0.5% v/v) as a vehicle was used as a negative control (scale bar = 100 μm; means ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 vs control [DMSO vehicle] unless indicated by bar). Refer to text for details.

Article Snippet: Mouse monoclonal anti-CA9 antibody (NBP1-51691) was from Novus Biologicals (Centennial, CO).

Techniques: Inhibition, Migration, Cell Culture, Control, Wound Healing Assay, Incubation, Negative Control

Journal: Redox Biology

Article Title: Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia

doi: 10.1016/j.redox.2019.101297

Figure Lengend Snippet: CA9 inhibition under hypoxia augments increase in catalytic Fe(II) with oxidative stress in mitochondria and lysosomes in MM cells. (A) Protein levels of CA9 and iron metabolism-associated proteins in ACC-Meso-1 cells after 48 h S4 treatment under hypoxia. (B) Levels of catalytic Fe(II) (SiRhoNox-1, 5 μM); (C) generation of mitochondrial O 2 − (MitoSox, 5 μM); (D) total cellular peroxides (CM-H2DCFDA, 5 μM) and (E) total lipid peroxidation (BODIPY, 2 μM) were quantified with FACS in response to S4 or U104 treatment (75 μM) under hypoxia for 48 h. (F) Localization of catalytic Fe(II) in S4-treated (75 μM) ACC-Meso-1 cells were determined by co-staining of SiRhoNox-1 (5 μM), LysoTracker (200 nM) and MitoTracker (75 nM) together after 48 h-hypoxic culture, followed by observation with a confocal microscope. DMSO as vehicle was used as negative control (0.5% v/v). Scale bar = 5 μm (N = 20; means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs control [DMSO vehicle]). Refer to text for details.

Article Snippet: Mouse monoclonal anti-CA9 antibody (NBP1-51691) was from Novus Biologicals (Centennial, CO).

Techniques: Inhibition, Staining, Microscopy, Negative Control, Control

Journal: Redox Biology

Article Title: Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia

doi: 10.1016/j.redox.2019.101297

Figure Lengend Snippet: CA9 inhibition induces mitochondrial fission and mitophagy in MM cells under hypoxia. (A) After treatment with S4 (75 μM) under hypoxia for 48 h, ACC-Meso-1 cells were stained by MitoTracker (200 nM) for 30 min, then fixed by 4% paraformaldehyde (PFA). Mitochondrial morphology was observed with a confocal microscope (scale bar = 10 μm). (B) The length of mitochondria was quantified with an ImageJ software (N = 20). Rel, relative. (C) Quantitative RT-PCR was performed to evaluate DRP1 mRNA level after treatment with S4 or U104 (40 μM) for 48 h under hypoxia by using GAPDH as internal standard. (D) Electron transmission microscope was used to examine the mitochondrial morphology after S4 treatment (75 μM) in ACC-Meso-1 cells for 48 h under hypoxia (bar = 1 μm). Red or white arrows represent mitochondrion or lysosome, respectively; AP represents autophagosome. (E) The same samples (shown in E) were used to demonstrate mitophagy, identified by co-staining of LysoTracker and MitoTracker in S4-treated (75 μM) ACC-Meso-1 cells (N = 20). (F) Immunofluorescent analyses were performed to confirm mitophagy. Cells were incubated with LC3 antibody (1:1000), following MitoTracker (200 nM) staining and fixation by 4% PFA (N = 10; scale bar = 5 μm). (G) Expression of LC3 and LAMP1 was evaluated in ACC-Meso-1 cells after treatment with S4 or U104 for 48 h under hypoxia (means ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 vs control [DMSO vehicle]). Refer to text for details. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Mouse monoclonal anti-CA9 antibody (NBP1-51691) was from Novus Biologicals (Centennial, CO).

Techniques: Inhibition, Staining, Microscopy, Software, Quantitative RT-PCR, Transmission Assay, Incubation, Expressing, Control

Journal: Redox Biology

Article Title: Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia

doi: 10.1016/j.redox.2019.101297

Figure Lengend Snippet: CA9 inhibition under hypoxia induced both apoptosis and ferroptosis in MM cells. (A) SYTOX Green staining of ACC-Meso-1 cells was performed to evaluate the effects of CA9 inhibitors (S4 and U104, 75 μM), which induced regulated cell death after 48 h-incubation under hypoxia (scale bar = 50 μm). Apoptosis was determined by detection of FITC-Annexin V with FACS (B) and TUNEL assay (C) . (D) Expression of cleaved caspase-3 in ACC-Meso-1 cells was accessed by western blotting after 48 h-treatment with S4 or U104 under hypoxia. (E) Effects of Fer-1 (3 μM), DFO (0.5 μM) and Z-VAD (50 μM) on S4 and U104 induced cell death (S4 50 μM or U104 60 μM) were evaluated after 72 h-treatment under hypoxia. (F) Effect of erastin (20 μM) on CA9 and iron metabolism-associated proteins and cell viability (G) in hypoxic ACC-Meso-1 cell (means ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 vs each control (CTRL) unless indicated by bar). Refer to text for details. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Mouse monoclonal anti-CA9 antibody (NBP1-51691) was from Novus Biologicals (Centennial, CO).

Techniques: Inhibition, Staining, Incubation, TUNEL Assay, Expressing, Western Blot, Control

Journal: ACS Nano

Article Title: Surface-Enhanced Raman Scattering Nanoparticles for Multiplexed Imaging of Bladder Cancer Tissue Permeability and Molecular Phenotype

doi: 10.1021/acsnano.8b03217

Figure Lengend Snippet: Schematic representation of the SERS nanoparticles developed herein. The Raman dyes were 4,4′-dipyridyl (s420, red dye), d8-4,4′-dipyridyl (s421, blue dye), and trans -1,2-bis(4-pyridyl)-ethylene (s440, green dye). The blue IgG4 nanoparticle is used as a negative experimental control for active binding of CA9- and CD47-targeted SERS nanoparticles.

Article Snippet: For negative controls the CA9 deactivating peptide (NB100-417PEP, Novus Biologicals) and a mouse IgG1 isotype (product no. MAB002, R&D Biosystems) for CD47 were used.

Techniques: Control, Binding Assay

Journal: ACS Nano

Article Title: Surface-Enhanced Raman Scattering Nanoparticles for Multiplexed Imaging of Bladder Cancer Tissue Permeability and Molecular Phenotype

doi: 10.1021/acsnano.8b03217

Figure Lengend Snippet: Validation of active targeting using flow cytometry. (a) Flow cytometry histogram showing binding of CD47-targeted SERS nanoparticles to cells with three negative controls. (b) Experiments from (a) were reported in triplicate and plotted in a bar graph. (c) Flow cytometry histogram showing binding of CA9-targeted SERS nanoparticles to cells with three negative controls. (d) Experiments from (c) were conducted in triplicate and plotted in a bar graph.

Article Snippet: For negative controls the CA9 deactivating peptide (NB100-417PEP, Novus Biologicals) and a mouse IgG1 isotype (product no. MAB002, R&D Biosystems) for CD47 were used.

Techniques: Flow Cytometry, Binding Assay

Journal: ACS Nano

Article Title: Surface-Enhanced Raman Scattering Nanoparticles for Multiplexed Imaging of Bladder Cancer Tissue Permeability and Molecular Phenotype

doi: 10.1021/acsnano.8b03217

Figure Lengend Snippet: Multiplexed imaging of CD47 and CA9 on cells in suspension. (a) Photograph of cells positive and negative for CD47 and CA9. The top right sample in the 2 × 2 grid is positive for CA9 but negative for CD47. (b) Spectral unmixing of background, s420-CA9, s421-IgG4, s440-CD47. The thick red line represents the fit, and the underlying thick dark blue line is the raw data. Thin lines represent the SERS and background components of the signal. (c) Overlay of the s440, s421, and s420 channel images on the photograph. (d) Calculated CD47:IgG4 and CA9:IgG4 ratio images overlaid on the photograph.

Article Snippet: For negative controls the CA9 deactivating peptide (NB100-417PEP, Novus Biologicals) and a mouse IgG1 isotype (product no. MAB002, R&D Biosystems) for CD47 were used.

Techniques: Imaging, Suspension

Journal: ACS Nano

Article Title: Surface-Enhanced Raman Scattering Nanoparticles for Multiplexed Imaging of Bladder Cancer Tissue Permeability and Molecular Phenotype

doi: 10.1021/acsnano.8b03217

Figure Lengend Snippet: Approach 1: Tissue classification based on raw, non-normalized SERS signal intensity. (a) Photograph and overlays of unmixed s420-CA9, s421-IgG4, and s440-CD47 images. (b) Stem plot of the non-normalized signal in each channel for all N = 15 samples. (c) Statistical comparison of the binding levels of s421-IgG4 in normal and tumor samples. (d) ROC plot for tissue classification using either all three channels (3-plex) or only the s421-IgG4 channel (passive). (e) Histological analysis of the data point indicated by a black asterisk in frame (b). Blue is DAPI nuclear stain, and red is a CA9 stain.

Article Snippet: For negative controls the CA9 deactivating peptide (NB100-417PEP, Novus Biologicals) and a mouse IgG1 isotype (product no. MAB002, R&D Biosystems) for CD47 were used.

Techniques: Comparison, Binding Assay, Staining

Journal: ACS Nano

Article Title: Surface-Enhanced Raman Scattering Nanoparticles for Multiplexed Imaging of Bladder Cancer Tissue Permeability and Molecular Phenotype

doi: 10.1021/acsnano.8b03217

Figure Lengend Snippet: Approach 2: Tissue classification based on active-to-passive normalized data. (a) Overlay CA9:IgG4 ratio on same tissue as Figure . (b) CD47:IgG4 ratio. (c) scatterplot of CD47:IgG4 and CA9:IgG4 ratios for N = 15 bladder tissue samples. (d) ROC curve for classifying bladder tissue based on 2-plexed and 3-plexed data. (e) SNR simulations of active-to-passive (Approach 2) and active-to-sum (Approach 3) ratios.

Article Snippet: For negative controls the CA9 deactivating peptide (NB100-417PEP, Novus Biologicals) and a mouse IgG1 isotype (product no. MAB002, R&D Biosystems) for CD47 were used.

Techniques:

Journal: ACS Nano

Article Title: Surface-Enhanced Raman Scattering Nanoparticles for Multiplexed Imaging of Bladder Cancer Tissue Permeability and Molecular Phenotype

doi: 10.1021/acsnano.8b03217

Figure Lengend Snippet: Approach 3: Classifying bladder tissue with active-to-sum normalized data. (a–c) CA9:sum, IgG4:sum, and CD47:sum overlays for the same tissue samples as in Figure a,b. (d) CD47:2-sum and CA9:2-sum ratios for all N = 15 tissue samples scanned on the endoscope. Because the 2-sum ratios only depend on either the s440-CD47 or s420-CA9 signal, the 2-sum ratios do not reflect correlations between s440-CD47 and s420-CA9 binding and thus fall directly on each axis. (e) CD47:3-sum and CA9:3-sum ratios for all N = 15 tissue samples. (f) ROC curves for classification of bladder tissue samples based on 3-plexed and 2-plexed active-to-sum ratios.

Article Snippet: For negative controls the CA9 deactivating peptide (NB100-417PEP, Novus Biologicals) and a mouse IgG1 isotype (product no. MAB002, R&D Biosystems) for CD47 were used.

Techniques: Binding Assay

Journal: ACS Nano

Article Title: Surface-Enhanced Raman Scattering Nanoparticles for Multiplexed Imaging of Bladder Cancer Tissue Permeability and Molecular Phenotype

doi: 10.1021/acsnano.8b03217

Figure Lengend Snippet: Summary of Signal Processing Approaches a

Article Snippet: For negative controls the CA9 deactivating peptide (NB100-417PEP, Novus Biologicals) and a mouse IgG1 isotype (product no. MAB002, R&D Biosystems) for CD47 were used.

Techniques: