Lung cancer search and destroy Like many cancer types, lung cancer is easier to treat when it is detected in its early stages, but this can be difficult to achieve. Scafoglio et al. discovered that a glucose transporter called sodium-glucose transporter 2 is not only expressed in lung cancer but also specifically found in early-stage tumors. Using a radiolabeled tracer specific for this receptor, the authors were able to perform positron emission tomography to identify early tumors that would otherwise remain undetectable. In addition, they found that a class of diabetes drugs called gliflozins, which target the same receptor, is effective at targeting these lung tumors in mouse models.

Abstract The diagnostic definition of indeterminate lung nodules as malignant or benign poses a major challenge for clinicians. We discovered a potential marker, the sodium-dependent glucose transporter 2 (SGLT2), whose activity identified metabolically active lung premalignancy and early-stage lung adenocarcinoma (LADC). We found that SGLT2 is expressed early in lung tumorigenesis and is found specifically in premalignant lesions and well-differentiated adenocarcinomas. SGLT2 activity could be detected in vivo by positron emission tomography (PET) with the tracer methyl 4-deoxy-4-[18F] fluoro-alpha-d-glucopyranoside (Me4FDG), which specifically detects SGLT activity. Using a combination of immunohistochemistry and Me4FDG PET, we identified high expression and functional activity of SGLT2 in lung premalignancy and early-stage/low-grade LADC. Furthermore, selective targeting of SGLT2 with FDA-approved small-molecule inhibitors, the gliflozins, greatly reduced tumor growth and prolonged survival in autochthonous mouse models and patient-derived xenografts of LADC. Targeting SGLT2 in lung tumors may intercept lung cancer progression at early stages of development by pairing Me4FDG PET imaging with therapy using SGLT2 inhibitors.

INTRODUCTION Non–small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Despite the success of targeted and immune-based therapies in NSCLC (1, 2), early diagnosis and surgical resection of early-stage disease remain the best opportunity for a cure: The 5-year survival of patients with NSCLC is 55.6% for localized disease but is only 4.5% for metastatic disease (3, 4). However, according to statistics of the National Institutes of Health (NIH) Surveillance, Epidemiology, and End Result Program, only 16% of newly diagnosed lung cancers are localized, whereas most has already spread to regional lymph nodes or to distant metastatic sites at the time of diagnosis (www.seer.cancer.gov/statfacts/html/lungb.html). Recently, intensive research efforts have been directed to the elucidation of the molecular mechanisms of pulmonary premalignancy development and progression (5–7) to find signatures of premalignancy that can be targeted for early diagnosis and cancer chemoprevention/interception (8, 9). Lung adenocarcinoma (LADC) and squamous cell carcinoma (SqCC) are the most frequent histologic subtypes of NSCLC. The sequence of premalignant lesions that leads to the development of SqCC has been extensively described and studied (10) because SqCC develops from the respiratory epithelium of large airways that are accessible by bronchoscopy. Conversely, LADC develops mostly within terminal bronchioles and alveoli, which are not readily accessible by bronchoscopy. Therefore, LADC premalignancy has been more elusive; the only premalignant lesion known to be a precursor of LADC is atypical adenomatous hyperplasia (AAH), consisting of a localized growth of premalignant, cuboidal cells lining the alveolar walls, defined as lepidic pattern (3, 11–14). AAH can progress to adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA), both of which are precursors of invasive adenocarcinoma and are also characterized by lepidic growth. AAH, AIS, and MIA can be detected in vivo by high-resolution computed tomography (CT), typically presenting as pure or predominantly ground-glass nodules (GGNs) (15–17). However, CT is not specific, and GGNs can also correspond to benign lesions, such as alveolar inflammation and hemorrhage. Thus, biomarkers to aid the diagnostic definition of GGNs and to identify AAH, AIS, and MIA noninvasively are urgently needed. An important hallmark of cancer is the increased requirement of glucose (18, 19). Glucose is thought to be imported into tumor cells primarily through specific glucose transporters known as GLUTs, which are frequently overexpressed in cancer cells (20–22). Glycolytic metabolism is readily imaged in cancer by positron emission tomography (PET) using a glucose analog, 2-deoxy-2-[18F] fluoro-d-glucose (FDG) (20). Although FDG PET is a standard tool for staging lung cancer, it has low sensitivity for identifying premalignant GGNs and early invasive disease (17, 23). The absence of FDG uptake in AAH and early-stage LADC has been interpreted as a consequence of a slow growth rate and low requirement of glucose (15, 17, 23, 24). However, we previously discovered that cancers can take up glucose by using an alternative glucose transport system, the sodium-dependent glucose transporters (SGLTs) (25, 26). SGLT-mediated glucose transport is not detected by FDG PET because FDG is not a transport substrate for SGLTs and is selective for only GLUTs (26, 27). Here, we present evidence that SGLT2 is a potential marker of pulmonary premalignancy and early LADC. SGLT-dependent glucose uptake can be selectively measured in vivo by PET imaging using the radiotracer methyl 4-deoxy-4-[18F] fluoro-alpha-d-glucopyranoside (Me4FDG). Me4FDG is a PET tracer transported specifically by SGLTs and not by GLUTs. This was demonstrated by in vitro studies showing that uptake of FDG can be completely abolished by GLUT inhibitor cytochalasin B and is not sensitive to SGLT inhibitor phlorizin (26). Conversely, Me4FDG uptake is completely abolished by phlorizin but is not affected by cytochalasin B, suggesting that Me4FDG cannot be transported by GLUTs (26). In vivo, Me4FDG, unlike FDG, cannot cross the blood-brain barrier because the capillary blood vessels of the blood-brain barrier express GLUTs but not SGLTs (27). In human PET scans, we have previously shown that Me4FDG gives no signal in the normal brain but is readily taken up by glioblastomas, which are typically characterized by microvascular proliferation, disruption of the blood-brain barrier, and SGLT2 expression (28). We previously showed SGLT2 expression in human pancreatic and prostate adenocarcinomas (25). Here, we demonstrate that LADCs predominantly use SGLT2 to transport glucose in the early stages of carcinogenesis. As tumors progress to advanced, poorly differentiated carcinomas, they up-regulate GLUT-mediated glucose transport, consistent with frequent positivity for FDG uptake. Using Me4FDG PET to image SGLT activity in vivo, we were able to functionally distinguish SGLT2-mediated active transport of glucose from GLUT1-mediated facilitative diffusion of glucose in genetically engineered murine models (GEMMs) and in patient-derived xenografts (PDXs) of lung cancer. Last, we showed that SGLT2 may be an effective therapeutic target in early-stage LADC in both GEMM and PDX models. Our results indicate that PET-guided imaging of SGLT2 activity may offer a means to identify metabolically active pulmonary premalignancy and early LADC and thus could guide targeted therapy/cancer interception with SGLT2 inhibitors.

DISCUSSION In this study, we present evidence that SGLT2 is a glucose transport mechanism specifically active in pulmonary premalignant lesions and in early LADC. We show the expression of SGLT2 protein by IHC with specific antibodies in human LADC specimens, in genetically engineered murine models and in PDXs of LADC. We also report SGLT2-dependent uptake of the PET tracer Me4FDG in vivo both in genetically engineered and in patient-derived murine models. Last, we confirm that SGLT2 inhibitors delay LADC development and growth in murine models, suggesting SGLT2 inhibition as a potential therapeutic strategy for premalignant and early-stage LADC. Our data highlight the importance of SGLT in early stages of LADC development, both in human tumors and in murine models. Our data suggest that there is a progression of glucose transporter expression during LADC carcinogenesis: Early-stage lesions express only SGLT2, whereas more advanced lesions display a spatially complex and heterogeneous pattern of SGLT2 and GLUT1 expression. Consistently, early-stage tumors accumulate only Me4FDG and not FDG, whereas advanced tumors take up both tracers. SGLT2 is most prominent in well-differentiated tumor cells, whereas GLUT1 becomes prevalent in poorly differentiated tumor cells. The mechanisms that drive this evolution in glucose transport in cancer are unknown and will need to be the subject of future investigations. However, we hypothesize that hypoxia is a major driver of the transition from SGLT2- to GLUT1-dependent glucose uptake in LADC. Hypoxia-inducible factor 1α up-regulates GLUT1 expression (40–42). We therefore speculate that premalignant and early-stage lesions, which are well oxygenated and perfused, preferentially express SGLT2. In contrast, it may be that, in advanced hypoxic lesions, GLUT1-mediated glucose transport dominates the tumor landscape. The discovery of SGLT2 expression in pulmonary premalignancy and early LADC has important diagnostic and therapeutic applications. The National Lung Screening Trial (NLST) has shown that low-dose helical CT in older, high-risk smokers can reduce lung cancer–related mortality by 20% compared with chest x-ray (43). However, CT imaging lacks specificity in distinguishing benign from malignant solitary pulmonary nodules (44). Moreover, high-resolution CT has increased the detection rates of indeterminate lung lesions (45), including both benign lesions and premalignant or early adenocarcinomas (46–48), which require additional imaging or invasive procedures for diagnosis. FDG PET has proven to be ineffective in identifying premalignancy or early LADC (17, 23), particularly in the setting of subsolid nodules (15–17) such as AAH, AIS, or MIA lesions (3, 14). Patients in the NLST with benign lesions (73%) received invasive diagnostic procedures (49). In addition to the costs and potential complications associated with these procedures, a quality-of-life (QOL) study on the impact of low-dose CT (LDCT) screening found that 46% of patients reported psychological distress while awaiting imaging results (50). This is particularly notable when considering that 16% of lesions identified on baseline screening LDCT in the NLST were ground-glass opacities that demonstrated relatively constant annual rates of lung cancer diagnosis over the course of 6 years of follow-up. Subsolid lung nodules commonly represent the early spectrum of LADC and may persist for years before malignant progression. Improved diagnostic tools could potentially reduce cost, invasive procedures, and radiation exposure, as well as improve QOL for patients with indeterminate lung nodules requiring surveillance. In addition to GGNs identified incidentally or by screening, it is common to detect multiple GGNs in patients who undergo surgery for an invasive LADC; Me4FDG PET could aid the follow-up of these lesions and help predict the risk of progression to invasive cancer. We present evidence that, in premalignant and early lepidic lesions, cellular glucose uptake occurs via SGLT2 rather than GLUT1 to satisfy metabolic demands. Therefore, it is anticipated that measurement of SGLT-mediated glucose utilization with the tracer Me4FDG will be valuable in classifying subsolid nodules as benign disease or early lesions within the spectrum of adenocarcinoma. This has the potential to answer an unmet need in the field of lung cancer that CT and FDG PET alone cannot address. Our next goal is to extend this work to clinical trials to evaluate Me4FDG for the diagnostic characterization of indeterminate lung nodules and GGNs and for predicting the clinical behavior of these lesions. We have also shown that SGLT2 inhibitors hinder tumor progression by limiting glucose supply in cancer cells and that Me4FDG can be used to evaluate the response of LADCs to SGLT2 inhibition by PET imaging before and after receiving treatment. Specific SGLT2 inhibitors (gliflozins), which are FDA approved for the treatment of diabetes, function by lowering the renal threshold for glucose reabsorption and therefore induce glycosuria and reduce blood glucose in patients with diabetes (35, 36). We have previously shown that gliflozins have antitumor activity against pancreatic tumors in a xenograft model (25). Here, we found that gliflozins specifically target lung premalignancy, effectively reduce tumor burden, and prolong survival if administered at an early stage. Our studies in PDX models treated with canagliflozin showed that Me4FDG uptake before treatment correlated with tumor volume fold decrease after treatment. This suggests that Me4FDG PET imaging could help identify individuals with premalignancy or early LADC with active SGLT2 transporters. In this context, gliflozin therapy could be applied as a cancer interception strategy to prevent or delay malignant progression of subsolid lesions detected by Me4FDG PET and CT (8, 9). This strategy would serve patients with other tobacco-associated cardiopulmonary diseases who are poor candidates for surgical resection. The ability to reliably detect premalignant and early stage LADCs with the use of Me4FDG PET could enable more timely interventions, interrupting the progression to invasive, more advanced disease and thus improving long-term outcomes. Last, we observed that Me4FDG PET, by detecting SGLT-dependent glucose transport in vivo, can be used to assess response to treatment with SGLT2 inhibitors. In particular, measurement of Me4FDG uptake in PDXs before and after beginning gliflozin treatment allowed us to establish an inverse correlation between the reduction in Me4FDG uptake as a consequence of the treatment and the rate of tumor volume increase. In patients with FDG-avid tumors, monitoring metabolic responses to drug therapy can be relevant for prognostic assessment and clinical decision making regarding treatment (51–53). Me4FDG PET in mice showed similar if not better minimal detection limits in early-stage lesions compared to FDG PET and would be expected to perform in an equivalent manner in humans (28). Therefore, we anticipate that PET measurement of SGLT activity in lung premalignancy and adenocarcinomas before and after the beginning of treatment will provide an invaluable precision medicine tool to evaluate the response of premalignant lesions to SGLT2 inhibitors. This study has some limitations. First, we were not able to demonstrate a significant correlation between SGLT2 expression and tumor stage. This is probably because of the low number of samples, especially considering that most surgical samples in the University of California, Los Angeles (UCLA) tumor bank were from early-stage tumors, with a relatively low number of advanced LADCs. However, we were able to find a specific profile of SGLT2 expression in lepidic and well-differentiated lesions. Second, we demonstrated Me4FDG uptake in GEMMs and in PDXs but we did not perform Me4FDG PET in patients with LADC; this will need to be the focus of future studies. Third, we cannot exclude that the effect of SGLT2 inhibitors on tumor volume and mouse survival is because of a systemic effect of lowering blood glucose rather than to the inhibition of glucose uptake in the tumor. Investigations using a conditional knockout of SGLT2 in the KP luc model should discriminate between these two possibilities.

MATERIALS AND METHODS Study design The purpose of this study was to evaluate SGLT2 as a diagnostic and therapeutic target for early-stage lung cancer. We validated this by IHC in human LADC specimens and by PET imaging and therapeutic trials in mouse models. For the IHC in human specimens, the purpose of the analysis was to assess the correlation between SGLT2 expression and tumor grade and stage. Samples of LADC were retrospectively selected from the UCLA lung tumor bank according to the pathologic grade and stage. The quantification of the signal was performed blindly by a board-certified pathologist (W.D.W.) using the Aperio ImageScope software. For the imaging and therapeutic trials in mouse models, we used a KrasG12D-driven, p53-null GEMM and PDXs of human LADC in nonobese diabetic (NOD), severe combined immunodeficiency (SCID), interleukin-2 receptor gamma knockout (NSG) mice. Mice were stratified to make the treatment groups comparable for mouse age (22.3 ± 0.54 weeks), sex (63% female, 47% male), body weight (33.5 ± 0.66 g), and tumor burden (estimated by bioluminescence signal in the GEMMs and by volumetric determinations from CT scans in PDXs). To determine the group size, we used a GEE model (54) to compare tumor size curves over time, assuming a two-sided 0.05 level of significance. With 12 mice per group, we calculated 86% power; therefore, for our therapeutic trials, we used groups of at least 12 mice. The tumor burden in the experimental groups was evaluated by objective measurements: (i) for the GEMMs, weekly bioluminescence measurements throughout the study; (ii) for the PDXs, measurement of tumor volumes from CT scans, performed by blindly designing ROIs encompassing the whole tumor volume; and (iii) measurement of tumor area by Definiens software in histologic lung sections stained with H&E. Mouse models All experiments performed in mice were approved by the UCLA Institutional Animal Care and Use Committee and were carried out according to the guidelines of the Department of Laboratory Animal Medicine at UCLA. All mice were housed in pathogen-free facilities at UCLA. Genetically engineered murine model. For our imaging and therapeutic trials in GEMMs, we used Lox-Stop-Lox KrasG12D, p53lox/lox, Rosa26-Lox-Stop-Lox-Luc mice previously established in our laboratory, inbred on a Friend virus B (FVB) background. The mice were bred in our colony at UCLA. Lung tumors were induced by intranasal administration of Adeno-Cre (purchased from University of Iowa Viral Vector Core) as previously described (30). After tumor induction, the tumor burden was estimated by weekly bioluminescence performed on an IVIS Spectrum In Vivo Imaging System (PerkinElmer) 10 min after intraperitoneal injection of luciferin (150 mg/kg). Patient-derived xenografts. The tumor samples were obtained from surgically resected specimens after informed consent under the UCLA Lung SPORE (PDX #004 and #011) or the Long Beach Memorial Hospital Institutional Review Board protocol (PDX #013). One of the PDXs (#186) was purchased from the Jackson laboratories. PDXs were established and passaged in NSG mice. NSG mice were procured from the UCLA Radiation Oncology breeding colony. For each mouse, two small pieces of tumor tissue (4 mm3) were implanted subcutaneously in the flank regions. MicroPET imaging For the PET imaging experiment in the GEMM presented in Fig. 2, the mice were scanned 12 weeks after the Adeno-Cre inhalation (1:200 dilution). For the time-course imaging in Fig. 3 (E to G), the mice received a much lower dilution of Adeno-Cre (1:10,000), and the mice were imaged when the average lung nodule maximum diameters were about 7 mm. For the microPET in PDXs, a subset of the mice (PDXs #004, #013, and #186) received both Me4FDG and FDG PET scans the day before and 2 weeks after the beginning of treatment to evaluate the response of glucose transporter activity to the treatment. The animals were anesthetized with 1.5% (v/v) isoflurane in oxygen, were given a dose of 100 μCi of Me4FDG or FDG via tail vein injection, and were maintained under anesthesia for 1 hour of unconscious uptake. The mice were then immobilized on the imaging bed and received a 10-min static PET scan followed by a CT scan. Each mouse received two different PET scans with the two tracers (Me4FDG and FDG) on consecutive days to allow for tracer decay. The equipments used were Focus 220 microPET scanner (Concorde Microsystems) and Inveon microPET scanner (Siemens) for the microPET scans and CrumpCAT (UCLA Crump Institute) for the microCT. The PET data were analyzed with AMIDE software version 1.0.4 (www.amide.sourceforge.net/) (55). ROIs for the measurement of tumor uptake were drawn corresponding with single lung nodules as identified by CT images. For the GEMMs, which typically present with small intrathoracic nodules with regular shape, ellipsoid ROIs were considered to be an acceptable approximation of tumor volume. For advanced lung nodules that did not have perfectly ellipsoid shape and smooth borders, the ROIs were placed in the center of the tumor nodule to include as much tumor volume as possible inside the ROI. To compare FDG and Me4FDG uptake, the same ROIs were used in the same mice scanned with the two different tracers, such that comparable tumor volumes were measured with the two tracers. For the PDXs, which typically show larger and irregularly shaped tumors, isocontour ROIs were designed on the basis of the CT scans to encompass the whole tumor volumes. The percentage of injected dose for each ROI was calculated by dividing the measured activity in the ROI by the total injected dose, as measured from the PET image by designing an ROI encompassing the whole mouse. The analysis of signal-to-noise ratio in the mouse nodules was performed as described in (31). Briefly, we evaluated the background signal for each mouse by designing a ROI corresponding to the normal lung (fig. S2D). We then calculated the CNR for each nodule by using the following formula (31) (1)where C l is the lesion to background contrast, n l is the number of pixels in the ROI, and SNR pixel is the signal-to-noise ratio for a single pixel in the background (2) The smallest lesion activity that can generate a CNR greater than 3 to 5 is called the minimum detectable activity. For the purpose of this analysis, we considered a CNR ≥ 4 as the specific signal. Therapeutic studies in mice Genetically engineered mouse model. Tumors were induced in KP luc mice by inhalation of Adeno-Cre (1:10 dilution). Two independent trials were performed starting 2 weeks after tumor induction. In the first trial (n = 12 mice per group), mice were treated for 6 weeks and then sacrificed for lung collection and fixation in formalin. In the second trial (n = 15 per group), mice were treated until death for survival analysis. The two therapeutic groups were as follows: (i) placebo, receiving daily oral gavage with vehicle (0.5% hydroxypropyl-methyl cellulose); and (ii) canagliflozin, receiving a daily dose of canagliflozin (30 mg/kg via oral gavage), as previously described (25). In the survival cohort, two mice (#3416 and #3418) were censored on days 45 and 57, respectively, for esophageal rupture because of complication of the oral gavage. Patient-derived xenografts. The mice were randomized in two therapeutic groups (same as for the trials in GEMMs): placebo and canagliflozin (30 mg/kg per day). The number of mice per group was four for PDX #011, eight for PDX #013, three for PDX #004, and five for PDX #186; each mouse was inoculated with two tumors (one tumor on each flank). Some of the tumors of PDX #011 developed soft tissue metastases in the axillary regions, and these were counted as separate tumors. Overall, 38 tumors were included in the placebo and 39 tumors in the canagliflozin group. The mice were treated for 1 month and then sacrificed, and the tumors were collected for histology and IHC. The mice of PDX #011 were treated only for 2 weeks because extremely rapid tumor growth in the placebo group required premature sacrifice of the animals. The tumor volumes at 2 weeks were counted as final tumor volumes. Immunohistochemistry For the GEMMs, the mouse lungs were collected and inflated with 10% formalin in phosphate-buffered saline and then incubated in formalin for 24 hours. For the PDXs, subcutaneous tumors were collected and incubated in formalin for 24 hours. All tissues were paraffin-embedded and sliced into 4-μm sections in the Translational Pathology Core Laboratory (TPCL) at UCLA. For human lung cancer samples, tissue blocks were obtained anonymously from the UCLA Lung SPORE tissue bank and from the Long Beach Memorial Hospital. For IHC staining, the slides were deparaffinized by overnight incubation at 65°C, followed by rehydration by serial passages in xylenes (three washes of 5 min in 100% xylenes) and decreasing concentrations of ethanol (two washes in 100% ethanol, two washes in 95%, one wash in 80%, one wash in 70%, and one wash in water). Antigen retrieval was performed for 20 min in 10 mM tris-HCl and 1 mM EDTA (pH 8.0) for SGLT2 and GLUT1 antibodies and in 10 mM citrate (pH 6.0) for Ki67. Blocking was performed with 5% goat serum for 1 hour at room temperature, followed by incubation with primary antibodies overnight at 4°C. Incubation with biotin-labeled secondary antibody was performed at room temperature for 1 hour, followed by incubation with avidin-biotin peroxidase complex (ABC; Vector Laboratories) and ImmPACT 3,3′-diaminobenzidine (Vector Laboratories) for 1 min. Counterstain was performed with Harris’ hematoxylin diluted 1:5 in water. For SGLT2, two different antibodies were used: Abcam ab85626 (1:1000) for mouse tissues and Novus Biologicals NBP1-92384 (1:250) for human and mouse tissues. The antigenic peptide for the Novus antibody is FHEVGGYSGLFDKYLGAATSLTVSEDPAVGNISSFCYRPRPDSYHLL; for the Abcam antibody, the sequence is proprietary but included in residues 250 to 350 of human SGLT2. For GLUT1, the Alpha Diagnostics GT11A antibody (1:200) was used. For Ki67, the Thermo Fisher Scientific SP6 antibody (1:200) was used. After the staining, digital images of the slides were obtained with an Aperio ScanScope slide scanner (Leica Biosystems). For the mouse tissues, the IHC signal was quantified with the Definiens Tissue Studio software. For the human tissues, the quantification was performed blindly by a board-certified pathologist (W.D.W.) using the Aperio ImageScope software. Statistical analyses The association between SGLT2 expression and Me4FDG uptake in mouse tissue was assessed using a GEE model (54) to accurately account for the same mouse being measured multiple times (multiple tumors per mouse or same mouse over time). We ran a similar model for testing the association between SGLT2 and GLUT1 expression. The associations were also quantified using Pearson’s correlation coefficient. The association between time (weeks 2, 5, and 8) and SGLT2 (or GLUT1) was formally assessed using the Jonckheere-Terpstra test for ordered alternatives. For the human samples, SGLT2 expression was computed using a weighted average, where each staining score assigned by the Aperio software (3+, very strong; 2+, strong; 2+, light; 1+, very light; 0, no signal) was multiplied by the corresponding percentage of cells in each sample. We then assessed the directional association between morphology categories (lepidic, moderately differentiated, and poorly differentiated) and SGLT2 or GLUT1 score using the Jonckheere-Terpstra test. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) v24. For the therapeutic trials in the GEMMs, tumor growth curves (slopes) were compared between canagliflozin and control groups using GEE models (54), with terms for time, group, and the time by group interaction. These models also included a random effect for mouse to account for the repeated observations of tumor size over time. We used log transformations to normalize the tumor size outcomes. For the therapeutic trials in PDXs, a linear mixed-effects model for log tumor volume was used, with terms for fixed effects (treatment group) and random effects (PDX trial; mice within trial clustered random effect: 38 to 39 distinct tumors in 20 distinct mice). The data analysis was performed using PROC MIXED from the Statistical Analysis Software (SAS) v9.4. All other group comparisons were performed using the two-sample t test unless otherwise noted. P values <0.05 were considered statistically significant throughout the manuscript.

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 KP luc mice. Fig. S3. Time course of PET/CT imaging with Me4FDG and FDG in KP luc mice. Fig. S4. Therapeutic trial with canagliflozin in KP luc 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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Acknowledgments: We thank J. Lee, C. Zamilpa, and T. Olafsen of the UCLA Crump Institute for Molecular Imaging for the constant support in performing the imaging experiments in mice and the personnel of the UCLA TPCL for the procurement of human samples and processing of tissues. We thank A. Fletcher for careful reading of the manuscript. Funding: C.R.S. was supported by the following grants: the NIH/National Center for Advancing Translational Science (NCATS) UCLA Clinical and Translational Science Institute KL2 Translational Science Award UL1TR001881, the Integrated Molecular, Cellular, and Imaging Characterization of Screen-Detected Lung Cancer NCI 1U01CA196408, the American Cancer Society Research Scholar Grant 130696-RSG-17-003-01-CCE, the Tobacco-Related Disease Research Program High Impact Research Project Award 2016TRDRP0IR00000143977, the STOP Cancer Foundation Seed Grant, and the Saul Brandman Foundation grant. D.B.S. was supported by a Department of Defense Lung Cancer Research Program grant W81XWH-13-1-0459. S.M.D. was supported by NCI EDRN 1U01CA214182, NCI 1U01CA196408, and Merit Review Research Funds from the Department of Veterans Affairs. J.Y. was supported by Thoracic Surgery Foundation Research Award and STOP Cancer I.C.O.N./Natasha Girard Seed Grant. B.V. was supported by the NIH/NHLBI training grant 5T32HL072752. Author contributions: D.B.S. and C.R.S. designed all the experiments, coordinated the work of all authors, and prepared the manuscript. C.R.S. performed most of the experiments, including mouse imaging, therapeutic trials, and IHC in mouse and patient tissues, and wrote the manuscript. B.V. performed IHC in PDX tissues and PET imaging in GEMMs and PDXs. G.A. contributed to the establishment, maintenance, and imaging of the PDXs and the imaging experiments. S.T.B. contributed to the establishment and maintenance of the genetically engineered mice. J.L. performed radiosynthesis of Me4FDG. A.S.S. facilitated the procurement of tissues. W.D.W. provided advice on the interpretation of clinical specimens and performed data analysis on human IHC samples. C.E.M. performed data analysis on all the mouse samples. T.R.G. and D.E. performed all the statistical analyses. J.R.B. contributed to the design of experiments and provided advice on the tracer synthesis and interpretation of the data. J.Y. and D.R.A. provided advice on the selection of patients and provided critical feedback on the manuscript. T.W. procured the human premalignancy specimens. S.M.D. provided advice and critical feedback on experiment planning and on the manuscript preparation. Competing interests: S.M.D. is on the Advisory Board of Early Diagnostics Inc., T Cure Therapeutics, and AstraZeneca. The tracer Me4FDG is covered by a UCLA patent application (E. M. Wright, J. R. Barrio, B. Hirayama, and V. Kepe, Tracers for monitoring the activity of sodium/glucose co-transporters in health and disease, U.S. Patent 8,845,999, application no. 11/920,904, filed on 23 May 2006). All other authors declare that they have no competing interests. Data and materials availability: All data associated with this study are present in the paper or the Supplementary Materials. The models presented will be available upon request to the corresponding authors after signing a material transfer agreement.