Extended Data Fig. 4: Differential response in oestrous cycle stages independent of T cell presence.

a,b, Representative immunohistochemistry staining and quantification of CD4⁺ T cells (a) and CD8⁺ T cells (b) in treatment naïve MMTV-PyMT mammary tumours during different stages of the oestrous cycle. n = 3 mice per oestrous stage and 2 tumours per animal. c, Schematic representation of the experimental setup. Mice were treated with depletion antibodies prior to doxorubicin treatment, which was initiated at a cumulative tumour mass of 500–750 mm³ in either oestrus (O) or dioestrus (D) stage. d, Representative immunohistochemistry staining of CD4⁺ T cells (top) and CD8⁺ T cells (bottom) in 36 tumours of 9 mice receiving T cell depletion antibodies (α-CD4 + α-CD8). e,f, Violin plots depicting percentage of dying tumour cells identified by positive staining for propidium iodide (e) and tumour volume change compared to baseline (f) for mice receiving T cell depletion antibodies (α-CD4 + α-CD8) and treated with doxorubicin. n = O^a-CD4/8 = 5 mice/20 tumours, D^a-CD4/8 = 4 mice/16 tumours. Data in a,b,e,f is depicted as violin plots, thick solid line represents median and solid lines indicate the 25th and 75th percentiles. Dots represent individual tumours (a,b,e) or mice (f). Scale bars, 500 µm (a,b,d). Statistical analysis was performed using linear mixed effects models (e), a two way anova (a,b) and a one-sided Wilcoxon-Mann-Whitney test (f). *P < 0.05. More details on statistical analysis can be found in Supplementary File 1.

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