Research ArticleCancer

Programmable probiotics for detection of cancer in urine

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Science Translational Medicine  27 May 2015:
Vol. 7, Issue 289, pp. 289ra84
DOI: 10.1126/scitranslmed.aaa3519
  • Fig. 1. PROP-Z probiotics for noninvasive cancer detection.

    The PROP-Z diagnostic platform is made up of probiotic EcN bacteria transformed with a dual-stabilized, high-expression lacZ vector as well as a genomically integrated luxCDABE cassette that allows for luminescent visualization without providing exogenous luciferin (blue). (1) PROP-Z is delivered orally. (2) Probiotics rapidly (within 24 hours) translocate across the gastrointestinal tract and (3) specifically amplify within metastatic tumors present in the liver. (4) PROP-Z expresses high levels of the enzyme lacZ (red), which can cleave systemically injected, cleavable substrates (green and yellow). Cleavage products of the substrates (yellow) filter through the renal system (5) into the urine for detection (6).

  • Fig. 2. Colonization of liver metastases by orally administered probiotics.

    (A) Representative healthy and tumor-bearing livers from Balb/c mice, excised ~21 days after intrasplenic injection of MC26-LucF cells bearing a firefly luciferase transgene. Arrow, small tumor nodules typical of those arising in this metastasis model. Scale bar, 5 mm. (B) Frequency distribution of tumor diameters in mice carrying MC26-LucF liver metastases 14 days after injection of the cells as measured by MRI. n = 10 mice, 98 tumors. (C) MRI images of a mouse with MC26-LucF liver metastases 14 and 17 days after injection of MC26-LucF cells. Yellow arrows, metastases. Scale bar, 10 mm. (D) Representative IVIS images for luminescence of MC26-LucF:Balb/c liver metastasis mice monitored over 21 days. (E) Average radiance for three mice shown as a function of time.

  • Fig. 3. Colonization of liver metastases by orally delivered PROP: Quantitation and specificity.

    (A) Twenty-four hours after oral delivery of PROP-Luc [5 × 109 colony-forming units (CFU)] to Balb/c mice carrying liver metastases (at 21 days), livers were excised and photographed (left), imaged for bacterial luminescence with IVIS (right), and then soaked in luciferin to visualize tumors with active mammalian MC26-LucF–derived luminescence (middle). Scale bars, 10 mm. (B) Distribution of colonized and uncolonized liver metastases from three mouse livers. Tumor diameters and colonization were determined by overlaying IVIS images, as described in Materials and Methods and fig. S2. (C) PROP-Z present in organs of healthy Balb/c mice (left) or liver metastases–bearing mice (right) after oral administration were quantified by traditional colony counting on erythromycin-LB plates and confirmed by a qPCR-based assay. Data represent mean colony counts from organs from n = 4 mice ± SEM. (D) Mice bearing DDC-induced liver injury (fibrotic damage visualized by a hematoxylin and eosin stain) were gavaged with 5 × 109 CFU PROP-Z. Excised livers were examined by IVIS. (E) Bacteria levels in liver metastases of control, DDC-treated animals, or Balb/c mice treated with MC26-LucF cells, as determined by colony counts (mean ± SEM; n = 5 each).

  • Fig. 4. Colonization in tumor models and different modes of administration.

    (A) IVIS images showing colonization of subcutaneous human (LS174T-LucF, TOV21G) or mouse (MC26, KBP22, 393M1-LucF) tumors by 5 × 106 CFU PROP-Z, 1 to 3 days after intravenous administration to immunocompetent mice. (B) Colonization was determined by observing flank luminescence from bacteria 1 to 3 days after bacteria administration using IVIS and is expressed as % tumors colonized. n = 6 tumors per cell line. (C) IVIS images of mice (MC26-LucF:Balb/c) intravenously injected with 104 or 5 × 106 CFU PROP-Z or administered with 5 × 109 CFU PROP-Z via oral gavage. (D) Colonization determined in two models upon oral (5 × 109) or intravenous (104 to 5 × 106) injection of PROP-Z and expressed as % colonized. n = 6 tumors per dosage.

  • Fig. 5. Dually stabilized vector efficiently maintains PROP-Z activity in vivo.

    (A) Bacteria luciferase signals from a constitutive luxCDABE circuit (PROP-Luc) or an AHL-inducible luxCDABE circuit (PROPi-Luc) 24 hours after bacterial intravenous injection. (B) The dual-stabilized maintenance system ensures plasmid stability in the tumor environment. (C and D) Bacteria were grown overnight with antibiotics (0 hours), and then subcultured without antibiotics and assayed for persistence of the lacZ plasmid by performing differential colony counts (black/white colonies on LB S-Gal plates) (see Materials and Methods) (C) and by assaying for lacZ enzymatic activity (D). au, arbitrary unit. Mean ± SEM; n = 4. (E) LacZ activity was assayed under reduced nutrient (glucose from 0.02 to 0.002%), pH, and oxygen in the presence of the lacZ inducer (1 mm of IPTG) or in noninducing conditions (0 mM). (F) The plasmid was measured after intravenous delivery of bacteria in a subcutaneous cancer model (LS174T-LucF, nude mice). Tumors were analyzed by colony counts on either plasmid-selective (kanamycin + erythromycin) or non–plasmid-selective plates (erythromycin only). Mean ± SEM; n = 5.

  • Fig. 6. Detection of metastatic tumors by PROP-Z urine diagnostic.

    (A) Subcutaneous tumor homogenates (nude mice, LS174T-LucF cell line) were analyzed for lacZ activity on days 0, 1, and 3 after intravenous injection of 5 × 106 CFU of PROP-Z bacteria (mean ± SEM; n = 5 each). (B) PROP-Z activity was quantified after systemic injection of LuGal, a lacZ substrate that when cleaved produces luciferin, which is measured with a subsequent urine assay for luminescence (mean ± SEM; n = 5, 6, and 9). P < 0.05 Student’s t test for the +bacteria/+liver metastasis/+lacZ case against the other two cases. (Inset) Representative cleavage assay using a color change substrate (CPRG) for the PROP-Z–treated mice either with or without liver metastases. (C) Paired measurements taken on days 0 and 1 after PROP-Z administered by gavage in individual mice. (Inset) Correlation of luciferase values on days 0 and 1 for each of the n = 10 mice in the cohort. (D) LacZ cleavage ratios shown for the 393M1-LucF liver metastasis model. Mean values ± SEM. P < 0.0001, one-way analysis of variance (ANOVA), Bonferroni post-test resulting in significant (P < 0.05) differences between day 0 versus day 2 and day 1 versus day 2. (Inset) IVIS image showing bacterial colonization of liver metastasis. (E) Urine assay lacZ cleavage data obtained after PROP-Z oral delivery to test animals (+liver metastasis, +PROP-Z), two cohorts of control mice [(M) mock surgery with PBS injected instead of PROP-Z; +liver metastases, +PROP–non-lacZ], and healthy mice (−liver metastases, +PROP-Z). (F) Receiver operating characteristic (ROC) plot illustrates the performance of a binary classifier system as the threshold is varied between case (+LM) and control (−LM) groups in data for (B). The area under the curve (AUC = 0.93) is a metric that characterizes the predictive power of the candidate diagnostic under these specific conditions.

  • Table 1. Bacterial strains and mammalian cell types used in this study.

    AHL, N-(3-oxohexanoyl)-l-homoserine lactone; GEMM, genetically engineered mouse model.

    LabelBacterial strain or
    mammalian cell
    Genomic/plasmidsUse(s)
    PROP-ZEcNluxCDABE (genomic),
    IPTG-inducible lacZ (stabilized plasmid)
    Bioluminescent imaging
    of bacteria, urine
    diagnostic assay
    PROP-LucEcNluxCDABE (genomic),
    luxCDABE (plasmid)
    Bioluminescent imaging
    of bacteria
    PROPi-LucEcNAHL-inducible
    luxCDABE (plasmid)
    Bioluminescent imaging
    of bacteria
    Non-lacZMach OnelacZΔM15 (genomic mutant)Non-lacZ control for urine
    diagnostic assay
    MC26-LucFMetastatic colorectal
    mouse cell line
    Firefly luciferase (genomic)Subcutaneous xenografts and
    liver metastasis models
    LS174T-LucFHuman colorectal
    adenocarcinoma cell line
    Firefly luciferase (genomic)Subcutaneous xenograft models
    393M1-LucFGEMM lung mouse cell lineFirefly luciferase (genomic)Subcutaneous xenografts and
    liver metastasis models

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/289/289ra84/DC1

    Fig. S1. Map of plasmid pTKW106 alp7A.

    Fig. S2. Size and colonization of liver metastases.

    Fig. S3. Calibration curve for the qPCR calculation of the number bacteria in a tumor sample.

    Fig. S4. Histopathological analysis of organs after oral delivery of PROP-Z.

    Fig. S5. Growth and activity of stabilized lacZ plasmid in different oxygen, inducer (IPTG), and antibiotic conditions.

    Fig. S6. CFU and stability of constructs in subcutaneous tumor models.

    Fig. S7. Background cleavage of LuGal from urine assay.

    Table S1. List and quantification of liver metastases.

    Table S2. Results of three-way factorial ANOVA.

  • Supplementary Material for:

    Programmable probiotics for detection of cancer in urine

    Tal Danino, Arthur Prindle, Gabriel A. Kwong, Matthew Skalak, Howard Li, Kaitlin Allen, Jeff Hasty, Sangeeta N. Bhatia*

    *Corresponding author. E-mail: sbhatia{at}mit.edu

    Published 27 May 2015, Sci. Transl. Med. 7, 289ra84 (2015)
    DOI: 10.1126/scitranslmed.aaa3519

    This PDF file includes:

    • Fig. S1. Map of plasmid pTKW106 alp7A.
    • Fig. S2. Size and colonization of liver metastases.
    • Fig. S3. Calibration curve for the qPCR calculation of the number bacteria in a tumor sample.
    • Fig. S4. Histopathological analysis of organs after oral delivery of PROP-Z.
    • Fig. S5. Growth and activity of stabilized lacZ plasmid in different oxygen, inducer (IPTG), and antibiotic conditions.
    • Fig. S6. CFU and stability of constructs in subcutaneous tumor models.
    • Fig. S7. Background cleavage of LuGal from urine assay.
    • Table S2. Results of three-way factorial ANOVA.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). List and quantification of liver metastases.

    [Download Table S1]

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