Fig 2: Schematic of pretargeting approach to image gastric tumors with pertuzumab in presence of lovastatin. Lovastatin depletes CAV1, increasing HER2 membrane availability and HER2 inactive dimers for binding pertuzumab. TCO-labeled pertuzumab (slow pharmacokinetics) is injected days ahead of administering radiolabeled small molecule. Then, only hours before imaging, administered radiolabeled small molecule travels through blood rapidly, either clicking with TCO-labeled antibody or quickly clearing from patient. HER2 is represented in light blue and CAV1 in yellow.

Fig 3: In vivo CAV1 modulation with statin improves HER2 stability at the cell membrane. a Membrane-bound and internalized [89Zr]Zr-DFO-trastuzumab before and after CAV1 depletion with 25 µM of lovastatin for 4 h in NCIN87 cancer cells. Increase in membrane-bound trastuzumab is rescued when cells are treated with lovastatin in the presence of 200 µM mevalonic acid (MVA). Treatment of NCIN87 cancer cells with a MßCD solution saturated with cholesterol increases [89Zr]Zr-DFO-trastuzumab internalization (bars, n = 5, mean ± S.E.M, **P < 0.01, ***P < 0.001 based on a Student’s t-test, n = 4). b Western blot of CAV1 in the total lysates of NCIN87 s.c. tumors from athymic nude mice. Lovastatin (8.3 mg kg-1 of mice) was orally administrated twice with an interval of 12 h between each administration. Tumor lysates were prepared between 0 and 48 h after the second dose of lovastatin and analyzed by western blot. c Treatment with lovastatin decreases HER2 shedding as determined by ELISA experiments (bars, n = 4, mean ± S.E.M, *P < 0.05 based on a Student’s t-test). d Fluorescence staining (HER2 and CAV1) and H&E stain of tissue sections of s.c. NCIN87 gastric tumors from athymic nude mice. Lovastatin (8.3 mg kg-1 of mice) was orally administrated twice with an interval of 12 h between each administration. Tumor sections were prepared at 48 h after the first dose of lovastatin. Scale bars: 100 µm. e, f Lovastatin increases cell membrane HER2 in NCIN87 tumors. Confocal images of immunofluorescence staining of HER2 in s.c. NCIN87 gastric tumors from athymic nude mice, non-treated (e) or treated with lovastatin (f). Images are two representative sections of two different xenografts. Lovastatin (8.3 mg kg-1 of mice) was orally administrated twice with an interval of 12 h between each administration. Tumor sections were prepared at 48 h after the first dose of lovastatin. Scale bars: 100 µm (left) and 50 µm (right)

Fig 4: Lovastatin enhances TDM1 efficacy and Trastuzumab-mediated ADCC.a–d Superior in vivo therapeutic efficacy of TDM1 combined with lovastatin when compared with TDM1 alone. a Intravenous TDM1 administration 5 mg/kg weekly (for 5 weeks) was started at day 0. Lovastatin (4.15 mg/kg of mice) was orally administrated 12 h prior to and simultaneously with the intravenous injection of TDM1. Lovastatin enhanced TDM1 efficacy of nu/nu female mice bearing NCIN87 gastric xenografts (b), and NSG mice bearing CAV1-high PDXs (d). *P < 0.05, **P < 0.01, ***P < 0.001 based on a Student’s t test (n = 8–10 mice per group). c Western blot analyses of AKT, ERK, Tyr, CAV1, and CREB protein expression and phosphorylation in NCIN87 xenografts at 40 days after treatment with lovastatin, TDM1, or TDM1/lovastatin. e NSG mice bearing NCIN87 xenografts were intravenously injected 1 × 106 human NK cells at day 0. One day after NK cells tail vein injection, the IL-15/IL-15Ra complex was intraperitoneally administered at a dose of 1.25 µg/mouse. Trastuzumab or Trastuzumab/lovastatin efficacy was then evaluated during a cytokine-dependent NK expansion phase (week 1–week 3). Lovastatin enhanced Trastuzumab efficacy in NSG mice humanized with NK cells and bearing NCIN87 xenografts (n = 8–10 mice per group, mean ± S.E.M.). Statistical analyses performed using ANOVA coupled to Scheffé's method. f Trastuzumab/lovastatin efficacy is higher than the combination of Fc-silent Trastuzumab (Trastuzumab F(ab’)2 fragments or deglycosylated Trastuzumab) in NSG mice humanized with NK cells and bearing NCIN87 xenografts (n = 8–10 mice per group, mean ± S.E.M.). Statistical analyses performed using ANOVA coupled to Scheffé's method. g Kaplan–Meier analysis of statin use and HER2-expressing GC disease outcome in patients treated with Trastuzumab. Patients without statin treatment (blue color, n = 27) have a worse survival than patients treated with statin (red color, n = 19). Log rank; p = 0.005. Source data are provided as a Source Data file.

Fig 5: Seesaw effect between CAV1 and HER2 proteins. a Association between CAV1 and HER2 in gastric (NCIN87, KATOIII), bladder (HT1197, UMUC14, UMUC3), breast (MDAMB231, MCF-7, BT474, SKBR3), pancreatic (BxPc3, MiaPaCa, Suit2) and prostate (LnCap, Pc3) cancer cells. The level of CAV1 (y-axis) and HER2 (x-axis) are expressed as the ratio of protein/ß-actin control, as determined by western blot analysis (upper panel). The non-parametric one-tailed Spearman test was used to determine the correlation coefficients. RHER2/CAV1, ratio of HER2-to-CAV1 protein levels as determined by western blot analysis from total extracts (lower panel). b Confocal images of immunofluorescence staining of HER2 in s.c. NCIN87 gastric tumors from athymic nude mice, showing that HER2 does not exhibit a predominant membrane staining. Scale bars: 100 µm and 50 µm (inset). c Confocal images of immunofluorescence staining of HER2 and CAV1 in s.c. NCIN87 gastric tumors from athymic nude mice. HER2 presence at the cell membrane in cells where CAV1 is absent (cell clusters 1 and 3) and HER2 almost absent at the cell membrane in cells where CAV1 is present (cell clusters 2 and 4). Scale bars: 50 µm. d Confocal images and quantification (mean ± S.E.M, n = 3) of immunofluorescence staining of HER2 and CAV1 in human HER2-positive gastric tumors containing high (Patient 1) and low (Patient 2) expression of CAV1. HER2 predominant presence at the cell membrane in CAV1-negative tumors (Patient 2). Scale bars: 50 µm. e Western blot analysis of the distribution of HER2 and CAV1 in caveolae fractions isolated from NCIN87 cells. f Western blot of HER2 and CAV1 in the total lysates of NCIN87 cells after blocking protein synthesis with 80 µg mL-1 CHX for 0, 12, 24, 30, 48, 60, 72, and 96 h. CHX cyclohexamide. g HER2 and CAV1 have similar half-lives. Half-lives calculated after western blot analysis of Fig. 1f. Density of western blot bands was quantified by scanning densitometry with ImageJ software. Half-lives were calculated as the time required for protein decrease to 50% of its initial level (mean ± S.E.M, n = 4)