Journal: Cell Death Discovery

Article Title: Macrophages produce and functionally respond to interleukin-34 in colon cancer

doi: 10.1038/s41420-020-00350-7

Figure Lengend Snippet: A , B Representative dotplots showing M-CSF1-R-expressing cells in TICs or lamina propria mononuclear cells (LPMCs) isolated, respectively, from tumoral and non-tumoral areas of surgical samples of one patient with colon rectal cancer (CRC). Cells were gated on live CD45 + cells and subsequently analyzed for the expression of M-CSF1-R and CD3 ( A ) or CD19 ( B ) by flow cytometry. One of five representative experiments is shown. Staining with the respective isotype control antibodies is also shown. C Representative dotplots showing M-CSF1-R-expressing cells in TICs or LPMCs isolated as above. Cells were gated on CD45 + live cells and subsequently analyzed for the expression of M-CSF1-R and CD68 by flow cytometry. The right panel shows live CD45 + CD68 + cells expressing M-CSF1-R in TICs or LPMCs isolated from five patients with CRC. Each point in the graph indicates the percentage of live CD45 + CD68 + cells expressing M-CSF1-R in a single sample of a single CRC patient. Horizontal bars indicate the median values. D Intestinal TICs were isolated from tumoral areas of five patients with CRC, and M-CSF1-R expression was analyzed in live CD45 + CD68 + HLA-DRII + cells by flow cytometry. Each point in the graph indicates the percentage of live CD45 + CD68 + HLA-DRII + cells either expressing or not M-CSF1-R in a single sample of a single CRC patient. Horizontal bars indicate the median values. Right panel: representative histograms showing the expression of M-CSF1-R in TICs isolated from tumoral area of one patient with CRC and analyzed by flow cytometry. The numbers indicate the percentage of live CD45 + CD68 + HLA-DRII + cells, either positive or negative, for M-CSF1-R. Histograms with isotype control antibodies are also shown.

Article Snippet: Slides were then incubated overnight at 4 °C with mouse anti-human IL-34 (final dilution 1:100, Abcam, Cambridge, UK), rabbit anti-human CD68 (final dilution 1:100, BioSpring Germany, Frankfurt).

Techniques: Expressing, Isolation, Flow Cytometry, Staining, Control

Journal: Cell Death Discovery

Article Title: Macrophages produce and functionally respond to interleukin-34 in colon cancer

doi: 10.1038/s41420-020-00350-7

Figure Lengend Snippet: A , B Representative dotplots showing IL-34-producing cells in TICs or lamina propria mononuclear cells (LPMCs) isolated, respectively, from the tumoral and non-tumoral areas of one patient with CRC. Cells were gated on live CD45 + cells and subsequently analyzed for the expression of IL-34, CD3 ( A ) or CD19 ( B ) by flow cytometry. One of five representative experiments is shown. Staining with the IL-34 isotype control antibodies is also shown. C Representative dotplots showing IL-34-producing CD68 + TICs or LPMCs isolated as above. Cells were gated on CD45 + live cells and subsequently analyzed for the expression of IL-34 and CD68 by flow cytometry. The right panel shows live CD45 + CD68 + cells expressing IL-34 in TICs or LPMCs isolated from five patients with CRC. Each point in the graph indicates the percentage of live CD45 + CD68 + cells expressing IL-34 in a single sample of a single CRC patient. Horizontal bars indicate the median values.

Article Snippet: Slides were then incubated overnight at 4 °C with mouse anti-human IL-34 (final dilution 1:100, Abcam, Cambridge, UK), rabbit anti-human CD68 (final dilution 1:100, BioSpring Germany, Frankfurt).

Techniques: Isolation, Expressing, Flow Cytometry, Staining, Control

Journal: Cell Death Discovery

Article Title: Macrophages produce and functionally respond to interleukin-34 in colon cancer

doi: 10.1038/s41420-020-00350-7

Figure Lengend Snippet: Representative images of double-immunofluorescence staining of colon sections taken from tumoral and non-tumoral areas of one patient with CRC and analyzed for the expression of IL-34 (green), CD68 (red), and DAPI (blue). The scale bars are 75 µm. Arrows indicate cells co-expressing IL-34 and CD68. Images at higher magnification (25 um) are also shown.

Article Snippet: Slides were then incubated overnight at 4 °C with mouse anti-human IL-34 (final dilution 1:100, Abcam, Cambridge, UK), rabbit anti-human CD68 (final dilution 1:100, BioSpring Germany, Frankfurt).

Techniques: Double Immunofluorescence Staining, Expressing

Journal: Cell Death Discovery

Article Title: Macrophages produce and functionally respond to interleukin-34 in colon cancer

doi: 10.1038/s41420-020-00350-7

Figure Lengend Snippet: A , B Intestinal TICs and LPMCs were isolated, respectively, from tumoral and non-tumoral samples of six patients with CRC and analyzed for the percentage of live CD45 + CD68 + HLA-DRII + IL-34+ cells either expressing ( A ) or not ( B ) M-CSF1-R + by flow cytometry. Each point in the graph indicates the percentage of positive cells in a single sample of a single CRC patient; horizontal bars indicate the median values. Right panels: representative histograms showing IL-34 expression in live CD45 + CD68 + HLA-DRII + TICs and LPMCs either expressing ( A ) or not ( B ) M-CSF1-R. Staining with isotype control antibodies is also shown. C Representative photomicrographs (×200 original magnification) of serial formalin-fixed, paraffin-embedded colon sections of surgical samples taken from the tumoral area of one patient with CRC and stained with IL-34 or CD68. The right panel shows the correlation between the number of IL-34-expressing cells and the number of CD68-positive cells as evaluated by immunostaining in frozen sections of surgical specimens taken from five CRC patients.

Article Snippet: Slides were then incubated overnight at 4 °C with mouse anti-human IL-34 (final dilution 1:100, Abcam, Cambridge, UK), rabbit anti-human CD68 (final dilution 1:100, BioSpring Germany, Frankfurt).

Techniques: Isolation, Expressing, Flow Cytometry, Staining, Control, Formalin-fixed Paraffin-Embedded, Immunostaining

Journal: Cell Death Discovery

Article Title: Macrophages produce and functionally respond to interleukin-34 in colon cancer

doi: 10.1038/s41420-020-00350-7

Figure Lengend Snippet: A , TICs ( A ) LPMCs ( B ) were either left unstimulated (Unst) or stimulated with increasing doses of human IL-34 (25–100 ng/ml) for 24 h, and the percentages of live CD45 + , CD68 + ,HLA-DRII + cells expressing CD163 and CD206 were analyzed by flow cytometry. Data are expressed as mean ± SEM of seven experiments. Right panels: representative dotplots showing CD163 and CD206 expression in live CD45 + CD68 + HLA-DRII + TICs ( A ) or LPMCs (B) isolated from respectively tumoral and non-tumoral samples of one patient with colon rectal cancer (CRC) and treated as described above.

Article Snippet: Slides were then incubated overnight at 4 °C with mouse anti-human IL-34 (final dilution 1:100, Abcam, Cambridge, UK), rabbit anti-human CD68 (final dilution 1:100, BioSpring Germany, Frankfurt).

Techniques: Expressing, Flow Cytometry, Isolation

Journal: Cell Death Discovery

Article Title: Macrophages produce and functionally respond to interleukin-34 in colon cancer

doi: 10.1038/s41420-020-00350-7

Figure Lengend Snippet: TICs ( A ) and LPMCs (B) were either left unstimulated (Unst) or stimulated with increasing doses of human IL-34 (25–100 ng/ml) for 6 h (left panel) or 48 h (right panel). Left panel: IL-6 RNA transcripts were analyzed by real-time PCR, levels were normalized to β-actin, and data are expressed as mean ± SEM of eight experiments. Right panel: IL-6 secretion was measured in the cell-free supernatants of TICs and LPMCs treated as above, and the data are expressed as mean ± SEM of eight experiments. C Intestinal TICs were isolated from tumoral samples of patients with CRC and transfected with a specific IL-34 antisense oligonucleotide (IL-34 AS) or with a scrambled antisense oligonucleotide (Src AS) (both used at 2 μg/ml) for 24 h. IL-34 and β-actin were analyzed by western blotting. One of three independent experiments is shown. D IL-6 secretion was measured in the supernatants of TICs isolated from tumoral samples of three patients with CRC and treated as described in C for 48 h. Horizontal bars indicate median values. E Intestinal TICs were isolated and treated as indicated in C and analyzed for the percentage of live CD45 + CD68 + HLA-DRII + CD163 + CD206 + cells expressing IL-6 (each point in the graph) by flow cytometry. Horizontal bars indicate the median values. Right panel: representative histogram showing IL-6 expression in live CD45 + CD68 + HLA-DRII + CD163 + CD206+ TICs isolated from tumoral samples of one patient with CRC, transfected with Src AS or IL-34 AS and analyzed by flow cytometry. Histogram with the respective isotype control antibody is also shown.

Article Snippet: Slides were then incubated overnight at 4 °C with mouse anti-human IL-34 (final dilution 1:100, Abcam, Cambridge, UK), rabbit anti-human CD68 (final dilution 1:100, BioSpring Germany, Frankfurt).

Techniques: Real-time Polymerase Chain Reaction, Isolation, Transfection, Western Blot, Expressing, Flow Cytometry, Control