Fig 2: Extracellular rCREG1 can reduce cell growth, migration, and invasiveness of PyMT cells. a PyMT cells treated with rCREG1 (400 nM) show reduced cell growth, migration, and invasiveness in real-time cell monitoring with an xCELLigence system. An effect observed representatively in the generated impedance curve (a; upper panel) and b quantitively by the slope ratio when compared to PBS-treated control (dashed line). c Closure of the gap experiments with live-cell imaging show reduced closure area in PyMT cells treated with rCREG1 (400 nM) when compared to PBS-treated control PyMT cells. ***p value ≤ 0.001; **p value ≤ 0.01; *p value ≤ 0.05; *Chi-square of linear mixed-effects model. wt wild type, rCREG1 recombinant murine CREG1

Fig 3: CREG1 changes upon Ctsb expression. a CREG1 abundance is increased in both co-cultures having Mϕ Ctsb−/−; Ctsz−/−, as well as in the TIF of PyMT+/0; Ctsb−/−; Ctsz−/− mice in MS proteomics studies. b Increased abundance is also observed by western blot using the CCM of co-cultures of tumor cell/macrophage deficient of both Ctsb and Ctsz and in co-cultures of wild-type tumor cells with Mϕ Ctsb−/−; Ctsz−/−. c In the TIF, the abundance of CREG1 changes upon Ctsb expression. CREG1 is increased in the TIF of PyMT+/0; Ctsb−/−; Ctsz+/+ mice, as well as in PyMT+/0; Ctsb−/−; Ctsz−/− mice, but decreased in the TIF with the transgenic expression of human CTSB (Tg(CTSB)+/0). CCM cell-conditioned media, TIF tumor interstitial fluid, Mϕ macrophage, wt wild type, dKO deficient from both Ctsb and Ctsz

Fig 4: Migration and invasiveness of PyMT cells can be modulated by CREG1. a PyMT cell 3D sphere cultures were generated in a methylcellulose/collagen I matrix show reduced sprout number and length when treated with rCREG1 (400 nM) for 24 h, representatively observed in spheroid images (upper left) and quantitively (upper right). b 3D spheres generated with PyMT cells with reduced Creg1 expression (shCreg1) show increased sprout number generation and length, detected representatively by spheroid images (lower left) and quantitively (lower right). **p value ≤ 0.01; ***p value ≤ 0.001. rCREG1 recombinant murine CREG1, shControl PyMT cell line with control shRNA, shCreg1 PyMT cell line with reduced Creg1 expression

Fig 5: CREG1 abundance in breast cancer tissue sections of the MMTV-PyMT model. a Immunostaining of CREG1 in breast tumor sections of PyMT+/0; Ctsb−/−; Ctsz−/− tumors shows stronger staining when compared to wild-type PyMT tumors, especially in stromal cells. A milder effect is seen in tumors from PyMT+/0; Ctsb−/−; Ctsz+/+ mice. No effect was observed in mice deficient for Ctsz. b Lysis of whole tumors shows an important increased abundance of CREG1 in tumors from PyMT+/0; Ctsb−/−; Ctsz+/+ and PyMT+/0; Ctsb−/−; Ctsz−/−, apparent at short exposure (upper lane). After long-exposure CREG1 is observed in tumors from wild-type mice (middle lane). Exp exposure