Kindlin-1 regulates IL-6 secretion and modulates the immune environment in breast cancer models
- Cancer Research UK Edinburgh Centre, Institute of Genetics and Cancer, University of Edinburgh, United Kingdom
Figures
Figure 1 with 2 supplements
Loss of Kindlin-1 leads to tumor clearance and immunological memory.
(A, C) Met-1 Kin1-WT, Kin1-NULL or Kin1-AA tumors were established via subcutaneous injection into flanks of CD1 nude mice (A) or FVB mice (C). Tumor growth was monitored and recorded until day 30, …
Figure 1—figure supplement 1
Loss of Kindlin-1 leads to reduction of tumor growth in human breast cancer model.
(A) Representative western blot demonstrating Kindlin-1 protein knockdown after addition of doxycycline to MDA-MB-231 cells expressing inducible non-targeting (NT) or FERMT1 shRNA. (B) …
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Figure 1—figure supplement 1—source data 1
Raw western blot images for Figure 1—figure supplement 1A, C.
- https://cdn.elifesciences.org/articles/85739/elife-85739-fig1-figsupp1-data1-v2.pdf
Figure 1—figure supplement 2
Loss of Kindlin-1 does not alter proliferation rate of Met-1 cells and tumors.
(A) Nanostring PanCancer panel of cell cycle genes in Met-1 cells. n=3 per group. (B, C) Immunohistochemical analysis of Ki67 (B) and phospho-histone H3 (C) in Met-1 tumors, with representative …
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Figure 1—figure supplement 2—source data 1
Nanostring PanCancer panel analysis of Met-1 Kin1-WT and NULL cells in vitro.
- https://cdn.elifesciences.org/articles/85739/elife-85739-fig1-figsupp2-data1-v2.xlsx
Figure 2 with 2 supplements
Loss of Kindlin-1 reduces tumor associated macrophages and increases cDC1 dendritic cells.
(A) Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Major myeloid populations were …
Figure 2—figure supplement 1
Flow cytometry gating examples of myeloid populations.
Example of tumor myeloid cell gating shown. First debris is removed by All cells gate, followed by singlets and live cells. CD45+ cells are selected for downstream identification of Macrophages …
Figure 2—figure supplement 2
Macrophage and dendritic cell profiling in Met-1 tumors and PD-L1- Kindlin-1 correlation in human breast cancer dataset.
(A) Quantification of MHC II and CD206 (left), MHC II and SIRPα (middle), and SIRPα and CD206 (right) as a percentage of total macrophages infiltrating MET-1 tumors. Data representative of two …
Figure 3 with 3 supplements
Loss of Kindlin-1 reduces tumor infiltrating Treg cells.
(A) Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Gating of major T cell populations was …
Figure 3—figure supplement 1
Flow cytometry gating examples of T cell populations.
Example of tumor T cell cell gating shown. First debris is removed by All cells gate, followed by singlets, live cells and lymphocytes. γδT cells (CD3+ γδTCR+) and T cells (CD3+ γδTCR-) were gated. …
Figure 3—figure supplement 2
Loss of Kindlin-1 reduces tumor infiltrating Treg cells.
(A) Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Gating of CD3+ cells was conducted …
Figure 3—figure supplement 3
Immune modulation in MMTV-PyV MMTV-Kin-1wt/wt and MMTV-Kin-1fl/fl spontaneous tumor model.
(A) Spontaneous MT-Kin-1wt/wt or MT-Kin-1fl/fl tumors were harvested once 1 tumour reached 10 mm for immunophenotyping by flow cytometry. Quantification of cDC1 cells as a percentage of total DCs …
Figure 4 with 1 supplement
Loss of Kindlin-1 modulates Treg phenotype and function.
(A) Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for RNA analysis of isolated CD45+ cells using Nanostring Immune Exhaustion …
Figure 4—figure supplement 1
Loss of Kindlin-1 modulates Treg phenotype markers.
(A–D) Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Analysis of expression of markers …
Figure 5 with 3 supplements
Loss of Kindlin-1 leads to altered cytokine secretion.
(A) Met-1 Kin1-WT or Kin1-NULL cells were cultured for 48 hr before conditioned media (CM) was harvested for analysis by forward phase protein array. Proteins detected above background are shown as …
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Figure 5—source data 1
Forward Phase Protein Array of Met-1 cells (raw values).
- https://cdn.elifesciences.org/articles/85739/elife-85739-fig5-data1-v2.xlsx
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Figure 5—source data 2
Nanostring PanCancer Immune panel analysis of Met-1 Kin1-WT and NULL cells in vitro.
- https://cdn.elifesciences.org/articles/85739/elife-85739-fig5-data2-v2.xlsx
Figure 5—figure supplement 1
Analysis of TGFβ signaling in Met-1 Kin1-WT, NULL tumors.
(A) RNA analysis of TGFβ signalling related genes in Met-1 Kin1-WT and NULL cells in vitro, using Nanostring PanCancer immune profiling panel. (B) Met-1 Kin1-WT, NULL and AA tumors were established …
Figure 5—figure supplement 2
Quantification of CXCL13 and IL-6 in Met-1 Kin1-WT, Kin1-NULL and Kin1-AA cells.
(A) Met-1 Kin1-WT, Kin1-NULL, and Kin1-AA cells were cultured for 48 hr before conditioned media (CM) was harvested for analysis by forward phase protein array. Data for CXCL13 and IL-6 from Figure 5…
Figure 5—figure supplement 3
Quantification of B cells in Met-1 Kin1-WT and Kin1-NULL tumors.
Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Gating of CD3- cells was conducted with …
Figure 6 with 1 supplement
Loss of Kindlin-1 leads to modulation of Treg differentiation and function.
(A–B) Met-1 Kin1-WT or Kin1-NULL cells were cultured for 48 hr before conditioned media (CM) was harvested. Naïve CD4+ T cells were isolated from FVB mice spleens and stimulated in the presence of …
Figure 6—figure supplement 1
Blocking CXCL13 does not alter Treg differentiation in vitro.
Met-1 Kin1-NULL cells were cultured for 48 hr before conditioned media (CM) was harvested. Naive CD4+ T cells were isolated from FVB mice spleens and stimulated in the presence of Kin1-NULL CM +/– …
Figure 7 with 1 supplement
Loss of tumor-derived IL-6 drives changes in Treg numbers and function but is not sufficient to reverse clearance of Kin1-NULL tumors.
(A) Met-1 Kin1-NULL IL-6-CTRL or Kin1-NULL IL-6-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Gating of CD4+ T …
Figure 7—figure supplement 1
Depletion of Tregs with anti-CD25 antibody treatment.
(A) After 3 day pre-treatment with anti-CD25 or isotype control antibody, Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice. Antibody treatment was continued …
Additional files
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Supplementary file 1
List of antibodies used for immunophenotyping.
- https://cdn.elifesciences.org/articles/85739/elife-85739-supp1-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/85739/elife-85739-mdarchecklist1-v2.docx
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Source data 1
NanoString PanCancer Immune panel analysis of bulk Met-1 tumors at day 10 (normalised expression), related to Figure 2 and Figure 5.
- https://cdn.elifesciences.org/articles/85739/elife-85739-data1-v2.csv
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Source data 2
Nanostring immune exhaustion panel of isolated CD45+ cells from Met-1 tumors at Day 10, related to Figure 2, Figure 4 and Figure 6.
- https://cdn.elifesciences.org/articles/85739/elife-85739-data2-v2.csv
