Fig 2: Loss of TRPV4 reduces tumor growth and cytokine expression known to activate hepatocytes and hepatic stellate cells.(A) Schematic of the orthotopic experiment. (B) Representative images of tumors from WT, Piezo1GFAP KO, or TRPV4-KO mice that were injected with 100,000 KPCY cells in the tail of the pancreas. Organs were collected 20 days after surgery. (C) Quantitative analysis of tumor size. (D–F) Serum levels of IL-6 (D), TIMP1 (E), and SAA (F) were measured by ELISA. (G) Saa2/Saa1 from liver RNA was measured by RT-PCR. Statistical analyses were performed using 1-way ANOVA with Dunnett’s post hoc test. Results are expressed as mean ± SEM.

Fig 3: The TRPV4 antagonist GSK2193874 protects mice from premetastatic niche formation.(A) Graphical illustration of the orthotopic surgery with daily gavage of GSK2193874 (10 mg/kg). (B) Quantitative analysis of tumor weight. (C and D) Serum cytokines levels IL-6 (C) and TIMP1 (D) were measured by ELISA. (E and F) Serum SAA was measured by ELISA (E) and liver expression of Saa1/Saa2 genes (F) by RT-PCR. Statistical analysis was performed using Student’s t test. Results were expressed as mean ± SEM. *P ≤ 0.05; **P ≤ 0.01.

Fig 4: Inflammatory mediator, CathK SP-A, and TIMP levels in WT and Trpml1−/− lung cells were measured using Multiplex and ELISA.(A–C) Quantification of the levels of different inflammatory mediators in BALF, macrophage, and pmLF supernatants (SN) isolated from WT and Trpml1−/− mice, using Multiplex (FirePlex) (A–C) as well as cathepsin K (CathK), surfactant protein A (SP-A), and TIMPs (D, E) using ELISA. BALF / pmLF SN: One single dot corresponds to one biologically independent sample i.e., one mouse. AMΦ SN: One single dot corresponds to one well. 8 WT and 8 Trpml1−/− mice were lavaged to obtain the appropriate number of cells for all wells. Statistical analysis of datasets in (A) was performed with multiple t-test, corrected for multiple comparisons using the Holm–Šídák method, ***p < 0.001. Data were mean ± SEM. Statistical analysis of datasets in (B, C) was performed using Student’s t-test, unpaired, two-tailed. (D) Transcriptomics data of single-cell suspensions from whole WT mouse lungs. Featured plot shows the average expression levels of TIMPs based on unique molecular identifier (UMI) counts (coded by color grading). TIMP expression was determined in 32 different cell types. Shown are TIMP1, 2, 3, 4. Source data are available online for this figure.

Fig 5: Characterization of the expression of additional MMPs in the murine lung using single-cell transcriptomics and qPCR/WB data of MMPs and TIMPs.(A) UMAP plot showing annotated cell clusters. Major cell types are labeled and color-coded, including epithelial, immune, endothelial, and stromal populations. MMP expression was determined in 32 different cell types. (B) UMAP plots showing the expression of selected MMP genes (MMP10, 11, 15, 16, 17, 20, 21, 23, 24, 25, 27, 28) in single cells from mouse lung tissue (filtered air group, n = 9). Expression is color-coded by log-normalized expression levels, with darker shades indicating higher expression. (C–E) qRT-PCR data showing mRNA expression levels of Mmp1a, Mmp1b, Mmp2, Mmp3, Mmp9, Mmp12, Mmp13, Mmp14, and Mmp19 in pmLF, IMΦ or AMΦ (WT and Trpml1−/−). (F) qRT-PCR data showing mRNA expression levels of Timp1, Timp2, Timp3, Timp4 in pmLF and IMΦ (WT and Trpml1−/−). In all figures, each single dot corresponds to one biologically independent sample. Data were mean ± SEM. Statistical analysis for qRT-PCR data were performed with multiple t-test, corrected for multiple comparisons using the Holm–Šídák method. (G–J) Western Blot analysis of different MMPs in pmLF isolated from WT and Trpml1−/− mice. Graphs show quantification of each MMP band normalized to ß-actin. Each single dot corresponds to cells isolated from one mouse. Data were mean ± SEM. Student’s t-test, unpaired, two-tailed. Source data are available online for this figure.