Extended Data Fig. 6: Neuronal substance-P drives breast cancer metastasis via the activation of the tumoral TACR1 receptor.
From: Neuronal substance P drives metastasis through an extracellular RNA–TLR7 axis
(a) Invasion assay of 67NR cancer cells in response to SP. *p = 0.0254, t-test. Mean ± SD. (b-c) 67NR cancer cell spheroids cultured with neuropeptides galanin or CGRP, with or without RNase A. (b) Invasion quantification. **p = 0.0011, ****p < 0.0001, nsp (CGRP) = 0.1066, nsp (galanin) > 0.9999, Kruskal-Wallis test. Vehicle-treated 67NR spheroids (−/+RNase A) are also plotted in Fig. 2d and Fig. 3d respectively since the experiments were conducted together. (c) Proliferation quantification. ***p = 0.0006, Mann-Whitney test. Mean ± SD. (d) ELISA-based quantification for SP levels in conditioned medium. p values are based on comparisons with base medium (NGM). nsp = 0.7426 (vs DRG only), 0.8724 (vs 67NR only); *p = 0.0213; **p = 0.0024, ANOVA. Mean ± SD. (e-g) In vivo measurements of SP expression. (e) Mean fluorescence intensity (MFI) of SP in 4T1 vs 67NR primary tumours. ****p < 0.0001, t-test. (f) Plasma SP levels in 67NR vs 4T1 tumour-bearing mice. *p = 0.0359, t-test. (g) MFI of SP in 4T1 vehicle vs capsaicin-treated primary tumours. ****p < 0.0001, t-test. Mean ± SD. (h-k) Effects of an SP-blocking antibody on 4T1 tumour growth and metastasis. (h) Schematic. (i) MFI of SP in IgG vs anti-SP treated 4T1 tumours. ****p < 0.0001, Mann-Whitney test. Mean ± SD. (j) Tumour growth. ****p < 0.0001, Mann-Whitney test. Mean ± SEM. (k) Metastatic area quantified by H&E. **p = 0.0012, t-test. Mean ± SD. (l-o) Orthotopic transplantation of EO771 LM2 cells into the abdominal mammary glands of Tac1-WT and Tac1-null host mice. (l) Schematic. (m) MFI of SP in primary tumours. ***p = 0.0003, t-test. Mean ± SD. (n) Tumour growth. ***p = 0.0003, t-test. Mean ± SEM. (o) Number of macro-metastases quantified by H&E. ***p = 0.0006, t-test. Mean ± SD. (p-r) Depletion of SP’s receptor, TACR1 in 4T1 cancer cell spheroids. (p) Validation of knockdown. ****p < 0.0001, ANOVA. MW: TACR1 (46 kDa). Quantification of spheroid invasion (q) and proliferation (r). ****p < 0.0001, Mann-Whitney test. Mean ± SD. (s-u) Orthotopic transplantation of 4T1 cancer cells depleted for TACR1. (s) Schematic. (t) Tumour growth. ****p < 0.0001, ANOVA. Mean ± SEM. (u) Metastatic area quantified by H&E. ***p = 0.0003, ANOVA. Mean ± SD.
