Research ArticleCancer

Sodium-glucose transporter 2 is a diagnostic and therapeutic target for early-stage lung adenocarcinoma

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Science Translational Medicine  14 Nov 2018:
Vol. 10, Issue 467, eaat5933
DOI: 10.1126/scitranslmed.aat5933
  • Fig. 1 SGLT2 is expressed in low-grade and GLUT1 in high-grade human LADC.

    IHC was performed in human LADC samples with antibodies specific for SGLT2 and GLUT1. (A) Invasive adenocarcinomas different disease grades, as indicated. (B) Quantification of SGLT2 (top) and GLUT1 (bottom) staining in different disease grades of LADC measured by Jonckheere-Terpstra test. ***P < 0.001. Scale bars, 50 μm. (C) Normal alveoli. (D) Premalignant lesions: atypical AAH, AIS, and MIA. Scale bars, 50 μm (inset, 25 μm). (E) Expression of SGLT2 and GLUT1 in different PDX models of LADC: well differentiated (PDX #004), moderately differentiated (PDXs #186 and #011), and poorly differentiated (PDX # 013). Scale bars, 50 μm.

  • Fig. 2 Glucose transporter expression and activity in LADC is heterogeneous.

    (A) KPluc mice were imaged with Me4FDG and FDG; the scatterplot reports the uptake of ROIs corresponding to single LADC nodules, expressed as percentage of injected dose per gram of tissue (% ID/g). The red dotted box highlights tumors with high Me4FDG and low FDG uptake. The correlation was calculated using Pearson’s coefficient (r). (B) Representative transverse sections of the PET/CT images show three distinct tumor nodules: t1, t2, and t3. Uptake of Me4FDG is detectable in small nodules (t2 and t3), which are FDG negative. (C) Pearson correlation between Me4FDG uptake in LADCs and SGLT2 protein expression as measured by IHC. The P value was calculated from generalized estimating equation (GEE) models (54). (D) Pearson correlation between SGLT2 and GLUT1 protein expression in ROIs corresponding to small areas of the tumor that were clearly identified in the hematoxylin and eosin (H&E) staining as purely low grade or high grade, as outlined in fig. S2F. The ROIs encompassing tumor areas with high-grade histology are reported as red circles, and the ROIs corresponding to low-grade tumor areas are represented by green triangles. (E) Representative images of SGLT2 and GLUT1 expression in low- and high-grade tumor areas. Scale bars, 100 μm (insets, 10 μm). (F) H&E stain in an adjacent section shows the different morphology of the low- and high-grade tumor areas. Scale bars, 100 μm (insets, 10 μm). (G) SGLT2 and GLUT1 protein expression was compared between low- and high-grade tumors using GEE models. ***P < 0.001. (H) Representative images of a human adenocarcinoma, adjacent slices of which were stained for SGLT2 (top) or GLUT1 (bottom). Scale bars, 1 mm. (I) Higher magnification of a well-differentiated (blue squares) and a poorly differentiated (red squares) area of the samples presented in (H). Scale bars, 100 μm.

  • Fig. 3 SGLT2 is expressed in early LADC and GLUT1 in advanced LADC in KPluc GEMMs.

    (A) Schematic representation and whole-lung H&E stain of LADC progression in KPluc mice at different time points after tumor induction, as indicated. Scale bar, 1 mm. (B) Time course of SGLT2 and GLUT1 expression in KPluc mouse lung lesions at different time points after tumor induction. Scale bar, 50 μm. (C and D) Quantification of the SGLT2 (C) and GLUT1 (D) IHC signal at different time points after tumor induction in KPluc mice, measured by Jonckheere-Terpstra test. *P ≤ 0.05 and ***P ≤ 0.01. (E) Time course of Me4FDG and FDG imaging in KPluc mice. The first time point was taken when tumor nodules reached an average diameter of 7 mm, and the subsequent time points were performed at 2-week intervals. (F and G) Quantification of Me4FDG (F) and FDG (G) signal in all seven mice included in the cohort (with a total of 16 tumors). n.s., not significant. (H and I) Schematic representation of glucose transporter expression in different stages of LADC: Premalignant lesions and early-stage LADC express only SGLT2 (H), whereas advanced tumors show spatial heterogeneity of glucose transport expression, with SGLT2 in well-differentiated and GLUT1 in poorly differentiated areas of the same tumor (I).

  • Fig. 4 SGLT2 inhibition delays development of LADCs in KPluc GEMMs.

    (A and B) The effect of single-dose SGLT2 inhibitor dapagliflozin on SGLT activity was evaluated by Me4FDG PET. (A) Representative 3D rendering of the PET/CT in the same KPluc mouse imaged on different days with Me4FDG without or with coinjection of dapagliflozin. (B) Comparison of Me4FDG uptake in n = 5 mice, expressed as percentage of injected dose per gram of tissue (% ID/g) in ROIs corresponding to single lung nodules (top) and to the bladder (bottom). Uptake was compared between groups using the two-sample t test. ****P < 0.0001 and **P < 0.01. (C) Schematic representation of the therapeutic trials. Adeno-Cre, adenovirally encoded Cre recombinase; AAH, atypical adenomatous hyperplasia; ADC, adenocarcinoma. (D to J) Therapeutic trial in KPluc mice treated with placebo or canagliflozin starting 2 weeks after tumor induction and carried on for either 6 weeks (week 8 cohort) or up to 3 months (survival cohort). (D) Representative images of bioluminescence in two mice belonging to the two treatment groups. (E) Quantification of the bioluminescent signal in the two therapeutic groups over time. The P value presented was from the interaction term between time/group from a GEE model (54). (F) Representative H&E stain of mouse lungs in the two treatment groups from both trials. Scale bar, 5 mm. (G) Quantification of the tumor area in the week 8 and survival cohorts. Tumor size was compared between placebo and canagliflozin groups using the two-sample t test. *P < 0.05. (H and I) Ki67 staining in premalignant lesions. (H) Representative images. (I) Quantification of the signal in the two groups. The groups were compared using t test. Scale bars, 50 μm. (J) Overall survival curves for each group were constructed using the Kaplan-Meier method and formally compared using the log-rank test.

  • Fig. 5 SGLT2 inhibition of Me4FDG positive LADC slows down tumor growth in PDXs.

    (A) Mice carrying PDXs were treated with canagliflozin (30 mg/kg) for 1 month. They were imaged with Me4FDG and FDG PET/CT 1 day before and 2 weeks after the beginning of treatment; in addition, the mice received weekly CT scans to monitor tumor growth. (B) Final tumor volumes on the last day of the trial, as measured by CT; the groups were compared using a linear mixed effects model for log (volume) with adjustment for trial and mouse random effects. (C to G) Results of the Me4FDG PET imaging in the mice included in the therapeutic trial. (C) Representative PET/CT images in transversal section of two mice, one in the placebo and one in the canagliflozin group, injected with Me4FDG before (day 0) and after (week 2) the beginning of the treatment. The Me4FDG scans presented are from the same two mice (white arrows, tumor; yellow arrowhead, bladder). (D) Quantification of Me4FDG uptake in the tumors, expressed as percentage of injected dose per gram of tissue (% ID/g), in all the mice included in the trial, measured by t test. ***P < 0.001 and **P < 0.01. (E) Correlation between pretreatment Me4FDG uptake in the tumors in the placebo group and the fold increase in volume from the beginning to the end of the therapeutic trial. Pearson’s correlation coefficient (r) and P value are reported. (F) Correlation between pretreatment Me4FDG uptake in the tumors in the canagliflozin group and the fold increase in volume from the beginning to the end of the therapeutic trial. Pearson’s correlation coefficient (r) and P value are reported. (G) Correlation between the percent change in Me4FDG uptake from the beginning to week 2 of the therapeutic trial and the fold increase in volume from the beginning to the end of the therapeutic trial. Pearson’s correlation coefficient (r) and P value are reported. (H to L) Results of the FDG PET imaging in the mice included in the therapeutic trial. The panels report the data relative to FDG uptake in the same mice presented in (C) to (G). (H) Representative PET/CT images in transversal section (white arrows, tumor). (I) Quantification of FDG uptake in the tumors. (J) Correlation between pretreatment FDG uptake in the tumors in the placebo group and the fold increase in volume. (K) Correlation between pretreatment FDG in the canagliflozin group and the fold increase in volume. (L) Correlation between the percent change in FDG uptake and the fold increase in volume from the beginning to the end of the therapeutic trial.

  • Table 1 Clinical characteristics of the patients with AAH included in the study.

    F, female; M, male.

    Patient #AgeSexSmoking statusPack yearsMeans of detectionPathological stageOverall stage
    158MCurrent40IncidentalT1aN0M0IA
    259MFormer60SymptomaticT3N0M0IIB
    354FNever0SymptomaticTisN0M00
    473FFormer31Screening chest X-rayT1aN0M0IA
    582FNever0IncidentalT1aN0M0IA
    675FFormer20IncidentalT3N0M0IIB
    7
    877MT1bN0M0IA
    970FFormer28SurveillanceT3N0M0IIB

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/467/eaat5933/DC1

    Fig. S1. IHC with SGLT2 antibody.

    Fig. S2. Quantification of tracer uptake and protein expression in lung tumors of KPluc mice.

    Fig. S3. Time course of PET/CT imaging with Me4FDG and FDG in KPluc mice.

    Fig. S4. Therapeutic trial with canagliflozin in KPluc mice.

    Fig. S5. Heterogeneity of morphology and glucose transporter expression in PDXs.

    Table S1. Clinical and pathological features of the 58 LADC samples included in the study.

    Table S2. Quantification of SGLT2 and GLUT1 expression in low- and high-grade LADC in GEMMs.

    Table S3. Time-course analysis of SGLT2 and GLUT1 expression in LADC in GEMMs.

    Table S4. BLI of the mice included in the SGLT2 inhibition trial (survival cohort).

    Table S5. BLI of the mice included in the SGLT2 inhibition trial (week 8 cohort).

  • This PDF file includes:

    • Fig. S1. IHC with SGLT2 antibody.
    • Fig. S2. Quantification of tracer uptake and protein expression in lung tumors of KPluc mice.
    • Fig. S3. Time course of PET/CT imaging with Me4FDG and FDG in KPluc mice.
    • Fig. S4. Therapeutic trial with canagliflozin in KPluc mice.
    • Fig. S5. Heterogeneity of morphology and glucose transporter expression in PDXs.
    • Table S1. Clinical and pathological features of the 58 LADC samples included in the study.
    • Table S2. Quantification of SGLT2 and GLUT1 expression in low- and high-grade LADC in GEMMs.
    • Table S3. Time-course analysis of SGLT2 and GLUT1 expression in LADC in GEMMs.
    • Table S4. BLI of the mice included in the SGLT2 inhibition trial (survival cohort).
    • Table S5. BLI of the mice included in the SGLT2 inhibition trial (week 8 cohort).

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