Fig 2: CRISPR‐Cas9‐generated formyl peptide receptor (FPR) 1 knockout (KO) A549 alveolar epithelial cells are protected from the detrimental effects of cigarette smoke extract (CSE) on epithelial wound repair. Using CRISPR‐Cas9, FPR1 KO and wild‐type cells were generated. Both cell lines are derived from a single cell. (A–C) DNA sequencing confirmed the FPR1 KO and showed a 11 base pair deletion on one chromosome and a one base pair insertion on the other chromosome. Both alterations led to the formation of a pre‐mature stop codon. (B) , P < 0.001. (D) FPR1 downregulation was confirmed using western blot. Representative blot of six independently performed western blots. (E) Transepithelial resistance measured in real‐time using the Electric Cell‐substrate Impedance Sensing (ECIS) system. Both FPR1 KO (black, ) and wild‐type (grey, ) A549 cells formed an epithelial barrier within 2 days. (F) Both FPR1 KO and wild‐type A549 cells were stimulated with 0% or 30% CSE for 1 h before being wounded using electroporation (30 s, 4500 μA). Epithelial repair was followed for 10 h after wounding. Wild‐type A549 cells stimulated with 30% CSE was significantly different from all three other groups, as tested by a two‐way analysis of variance (ANOVA). *P < 0.05. All ECIS experiments were repeated eight times (, FPR1 KO; , wild‐type; , FPR1 KO + 30% CSE; , wild‐type + 30% CSE).

Fig 3: Tumor growth in nude mice under hypoxic condition. Inhibition of FPR1 activity suppressed the hypoxia-induced increase in (A) tumor volume and (B) tumor weight. (C) Inhibition of FPR1 activity suppressed the proliferation of tumor cells. Asterisks indicate statistical significance (***, P<0.001). Data are presented as the mean ± SD (experiments, n=3). Scale bar =100 µm. FPR1, formyl peptide receptor 1; SD, standard deviation.

Fig 4: (A) Expression of FPR1 in U87-MG cells, cultured as monolayers, in flow cytometry over isotype control, under normal cell culture condition, serum starvation, and hypoxia (* < 0.05, ** < 0.005, *** < 0.0005, **** < 0.0001). (B,D) Expression of FPR1 by IHC in sections from a U87-MG multicellular spheroid (bar = 200 micron, 100 micron and 50 micron respectively). Basophilic structure of cell was stained in blue with haematoxylin solution. (E) Expression of FPR1 by IHC in sections from a U87-MG xenotransplanted tumour tissue.

Fig 5: Transcriptome analysis (and selected transcript and expression validation) of noninvaded versus invaded breast cancer cells in CIMMS.(A) Read counts were collected and averaged from invaded and noninvaded subpopulations of MDA-MB-231 cells harvested from five simple collagen I microgels (2.4 mg/ml) on day 4 after seeding at 400,000 cells/ml. The data are represented as a heat map of expression level (shown as variance-stabilized read counts per million, where yellow = high and blue = low) of the 244 genes (rows) determined to be significantly differentially expressed [with FDR < 0.05 based on the quasi-likelihood negative binomial generalized log-linear model (glmQLFit) from the edgeR package (51)] for noninvaded (“N avg.,” left column) and invaded cells (“I avg.,” right column). The white trace labeled “density” in the legend represents the distribution of the different expression levels in the dataset. Four genes (FPR1, BCL3, CX3CL1, and CDH1) are highlighted; the full list is found in table S1 (list 1). (B) ddPCR results for transcripts of FPR1 (top), BCL3 (mid-top), CX3CL1 (mid-bottom), and CDH1 (bottom) in noninvading (white bars) and invading (black bars) cells harvested from CIMMS experiments treated identically to (A), with error bars = 1 SD. Expression values for the four genes were normalized to the expression level of housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH). (C) Top-view fluorescent confocal microscope images of microgels from CIMMS experiments treated identically to (A), with images in the left column stained blue for nuclei, images in the middle column immunostained green for FPR1 (top), BCL3 (mid-top), CX3CL1 (mid-bottom), and E-cadherin (bottom), and images in the right column overlaid. The white dashed lines indicate the edge of the gels. Scale bars, 50 μm.