Understanding of the factors governing immune responses in cancer remains incomplete, limiting patient benefit. In this study, we used mass cytometry to define the systemic immune landscape in response to tumor development across five tissues in eight mouse tumor models. Systemic immunity was dramatically altered across models and time, with consistent findings in the peripheral blood of patients with breast cancer. Changes in peripheral tissues differed from those in the tumor microenvironment. Mice with tumor-experienced immune systems mounted dampened responses to orthogonal challenges, including reduced T cell activation during viral or bacterial infection. Antigen-presenting cells (APCs) mounted weaker responses in this context, whereas promoting APC activation rescued T cell activity. Systemic immune changes were reversed with surgical tumor resection, and many were prevented by interleukin-1 or granulocyte colony-stimulating factor blockade, revealing remarkable plasticity in the systemic immune state. These results demonstrate that tumor development dynamically reshapes the composition and function of the immune macroenvironment.
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All mass cytometry data are publicly available at https://premium.cytobank.org/cytobank/projects/2433/ or by request to the senior author without restrictions.
The updated Statistical Scaffold package is available at https://github.com/SpitzerLab/statisticalScaffold.
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We thank the UCSF Flow Cytometry Core and S. Tamaki for CyTOF maintenance and M.H. Barcellos-Hoff, R. Levine, H. Okada, E. Engleman and J. Bluestone for cell lines, transgenic mice and reagents. We thank L. Lanier, Z. Werb, M.H. Barcellos-Hoff and L. Fong for insightful feedback. This work was supported by National Institutes of Health (NIH) grants DP5OD023056 and P50CA097257 (UCSF Brain Tumor SPORE Developmental Research Program), funds from the UCSF Program for Breakthrough Biomedical Research and investigator funding from the Parker Institute for Cancer Immunotherapy to M.H.S. and by NIH grant S10OD018040, which enabled procurement of the mass cytometer used in this study. This study makes use of data generated by the Norwegian Women and Cancer Study. A full list of investigators who contributed to the generation of the data is available at http://site.uit.no/nowac/. Funding for the project was provided by European Research Council grant ERC-2008-AdG 232997. The Norwegian Women and Cancer Study group is not responsible for the analysis or interpretation of the data presented.
M.H.S. receives research funding from Roche/Genentech, Bristol-Myers Squibb and Valitor and has been a paid consultant for Five Prime Therapeutics, Ono Pharmaceutical and January Inc.
Peer review information Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Main gating strategy for identifying major immune cell populations from mass cytometry datasets.
a, Relative abundance of total leukocytes infiltrating the TME across eight tumor models. b-f, Scaffold maps of spleen cell frequencies across five distinct tumor models, SB28 glioblastoma (b), MC38 colorectal (c), LMP pancreatic (d), B16 melanoma (e), and Braf/PTEN melanoma (f), comparing late stage tumor burden to their respective health littermate controls. g, Heatmaps of the log2 adjusted fold change in bulk immune cell frequencies across all five tissues, where relevant, across all models. h, Heatmaps of the log2 adjusted fold change in bulk immune cell frequencies comparing the parental AT3 and engineered AT3 expressing reporters GFP and Luciferase, with cell labels in g. Lower inset shows Pearson’s correlation between these systemic immune features.
a, Pearson’s correlation between MMTV-PyMT primary tumor size and change in systemic immune composition, measured as Aitchison distance. b, Degree of systemic immune change by Aitchison distance over tumor growth (left) and after removing the contribution of primary tumor size by linear regression (right). c, Percent of PyMT expressing metastatic cancer cells in the lung (green) and primary draining lymph node (blue). d, Pearson’s correlation between lung or lymph node metastasis and the residual changes in systemic immune composition after regressing out primary tumor burden. e, Heatmap of the log2 adjusted fold change in bulk spleen immune cell frequencies for each 400mm2 tumor-bearing mouse, ranging from 0 to high metastatic disease. f, Pearson’s correlation between tumor mass and absolute number of infiltrating leukocytes in 4T1 breast tumors. g, Spleen immune absolute cell counts, adjusted absolute cell counts per mg of tissue, and unadjusted immune frequencies at each time point for neutrophils, B cells and T cells of the 4T1 breast tumor model. h, PCA of relative immune cell frequencies from each major immune tissue over time in the MMTV-PyMT breast tumor model. Vectors designate progression from control (first point) to 25 mm2, 50mm2, 125mm2, and 400mm2 (last point, arrowhead). i, Scaffold maps of immune cell frequencies in the spleen at each time point of 4T1 tumor burden, colored by log2 fold change in frequency compared to the previous time point.
a-d, Scaffold maps of immune cell frequencies over 4T1 tumor progression in the tumor draining lymph node (a) blood (b), bone marrow (c), and tumor (d), colored by fold change from the previous time point.
a-b, PCA of T cell cluster frequencies across lymphoid tissues over tumor development for the 4T1 (a) and MMTV-PyMT (b) breast tumor models. Vectors designate directional progression from control (first point) to late stage disease (last point, arrowhead). In a, tumor time points include day 7, 14, 21, and 35 after 4T1 cancer cell transplant. In b, tumor time points include tumor sizes of 25 mm2, 50 mm2, 125 mm2, and 400 mm2. c-e, CD3 + CD11b- leukocytes from all tissues clustered together from healthy and MMTV-PyMT tumor-burdened animals at progressive tumor sizes. c, Heatmap of each T cell cluster frequency, by row, in each site and across the individual 2-3 animals per time point. d, Stacked bar plot of the log2 fold change in cluster frequency between early (25 mm2) and late (400 mm2) disease time points, colored by tissue. e, Heatmap of the protein expression defining each T cell cluster, column normalized to each protein’s maximum positive expression. f-h, Representative scatter plots of key proteins that define T cell clusters changing in frequency in the designated site between early and late disease stage for CD8 T cells (f), Tregs (g), and CD4 T Cells (h).
a, CD3- CD19- leukocytes from all tissues clustered together from healthy and 4T1 tumor-burdened animals at progressive time points. Left, stacked bar plot of the log2 fold change in cluster frequency between early (day 7) and late (day 35) times points, colored by tissue. Right, heatmap of the protein expression defining each cluster, column normalized to each protein’s maximum positive expression. b, Curves of the mean cell frequencies over time in the 4T1 breast tumor model from designated mononuclear phagocyte cell types, colored by tissue. c, PCA of the mononuclear phagocyte cell frequencies from each tissue over time in the 4T1 breast tumor model. Vectors designate progression from control (first point) to day 7, 14, 21, and 35 (last point, arrowhead). Coloring of tissues for a-c corresponds to labels in c.
a, Distribution of PD-1 and PD-L1 signal intensities on tumor infiltrating leukocytes over time in the 4T1 or AT3 breast tumor models. Coloring of time points for a-d corresponds to legend in a. b, Percent of total infiltrating leukocytes (left of dashed line) or CD45−, non-endothelial cells (right of dashed line) with high PD-1 or PD-L1 expression in the 4T1 or AT3 tumor models. c, Percent of leukocytes with high PD-1 or PD-L1 expression over time and across tissues, 4T1 model. d, Pearson’s correlation between median PD-L1 signal intensity on blood versus tumor infiltrating leukocytes, 4T1 model. e, Percent of each major immune cell subset expressing high PD-1 or PD-L1 in the tumor, blood, and spleen, identified manually. Cell subsets below 0.2% of total leukocytes were not included, X. Bars ordered by time point, beginning at healthy control. Double positive PD-1/PD-L1 expression was rare and not illustrated. p*< 0.05, One-Way ANOVA, with Tukey correction versus control tissue or healthy mammary fat pad (blue in b-c, fill corresponding to bar color in e), or versus day 7 (green in b-c).
a-b, Log2 fold change in bulk Ki67 expressing leukocytes in each tissue tissues for 4T1, AT3 and MMTV breast tumors (a), and over 4T1 tumor progression (b). p*< 0.05, One-Way ANOVA, with Tukey correction versus control. c-d, Statistical Scaffold maps of Ki67 expression in immune cells of the tumor draining lymph node comparing control to day 21 (c) and the Spleen over time (d) in 4T1 tumor burdened animals. e, Percent of increasing clusters (red, total of 56), decreasing clusters (blue, total of 90), or unchanged cluster that have corresponding changes in cell cycle markers Ki67 and cleaved Caspase-3.
a, Quantification of bulk CD8+ T cell populations in the spleen of healthy or AT3 tumor-burdened mice after 7 days of Lm infection, Two-Way ANOVA with Bonferroni correction. b, Expression of inflammatory cytokines, INFy, IL-2, and TNFa in splenic CD8 T Cells isolated from control or AT3 tumor-burdened mice after in vitro differentiation with CD3, CD28 and IL-2, and re-stimulation with BrefeldinA and PMA Ionomycin. c, Scatter plots of CD11b and Ly6G showing expected neutrophilia in OT-I TCR transgenic mice with AT3 tumor burden. d, Histograms of CD80, CD86, and CD83 signal intensity on cDCs from healthy or AT3 tumor-burdened mice at day 2 of Lm-OVA infection. e, Median signal intensity of CD80, PD-L1 and CD54 activation markers on splenic cDCs from healthy or AT3 tumor-burdened mice compared to IL-12p70 or CD40 treatment at day 7 of Lm-OVA infection. F, Median signal intensity of PD-L1 on splenic cDCs from untreated or CD40 treated AT3 tumor-burdened (day 21) mice. G, Quantification of splenic CD8 + T cell proliferation in healthy, untreated or CTLA-4 treated AT3 tumor-burdened animals in response to 7 days of Lm-OVA infection. p*<0.05, two-tailed t-test.
a-c, Statistical scaffold maps of spleen immune cell frequencies (a) and proliferation by Ki67 expression (b) in 4T1 resected mice, and of spleen immune cell frequencies in MC38 resected mice (c) compared to healthy control. Insets show resected mice compared to tumor-burdened mice. d-e, Heatmap of the log2 fold changes in splenic immune cell frequencies for local or lung recurrences from control mice (d), and for IL-1, G-CSF, or TGFβ blockade from untreated AT3 tumor-burdened mice (e). f-g, Heatmaps of T cell cluster expression profiles and log2 fold change from control for AT3 (f) and 4T1 (g) for the spleen and draining lymph node. h, Median signal intensity of CD86 and PD-L1 on splenic cDCs from healthy, AT3 tumor-burdened, resected, or resected mice with local recurrence at day 7 of Lm-OVA infection. i, Concentration of circulating cytokines, IL-1α and G-CSF from healthy, AT3 tumor-burdened, resected, or resected mice with local recurrence. j, Concentration of cytokines, IL-1α, IL-1β and G-CSF from in vitro cell culture media conditioned with AT3 cancer cells. k, Concentration of circulating G-CSF from control or AT3 tumor-bearing mice, or AT3 tumor-bearing mice treated with either IL-1 or G-CSF blocking antibodies. p*<0.05, two-tailed t-test.
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Allen, B.M., Hiam, K.J., Burnett, C.E. et al. Systemic dysfunction and plasticity of the immune macroenvironment in cancer models. Nat Med 26, 1125–1134 (2020). https://doi.org/10.1038/s41591-020-0892-6
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