Research ArticleCELL ENGINEERING

Synthetic biology-based cellular biomedical tattoo for detection of hypercalcemia associated with cancer

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Science Translational Medicine  18 Apr 2018:
Vol. 10, Issue 437, eaap8562
DOI: 10.1126/scitranslmed.aap8562
  • Fig. 1 Design and optimization of the cell-based biomedical tattoo system.

    (A) Molecular mechanism of pigment production by HEKTattoo cells upon induction by hypercalcemia. The sensing component of the biomedical tattoo system (I) is a calcium-sensing receptor (CaSR), a member of C group G protein–coupled receptors. The visualization component (II) consists of the enzyme tyrosinase, which is usually located in a specialized pigment-producing organelle (the melanosome) in melanocytes, where it catalyzes oxidation of phenols such as tyrosine to form melanin. In nonmelanogenic cells such as human embryonic kidney–293 (HEK-293) cells, tyrosinase is localized in lysosomes, which are closely related to melanosomes and undergo a similar maturation process. IP3, inositol 1,4,5-trisphosphate; NFAT, nuclear factor of activated T cells; SRF, serum response factor. (B) Secreted alkaline phosphatase (SEAP) expression in response to calcium stimulation in HEK-293 cells transiently transfected with a constitutive CaSR construct (pAT12, PhCMV-CaSR-pA) and either an inducible SEAP reporter construct (pAT16, PCa4-SEAP-pA) or a mock construct [pcDNA3.1(+)]. Increase in SEAP expression compared between 0.5 and 1.8 mM Ca2+ concentration, P = 0.026, determined using independent t test where *P < 0.05. (C) Optimization of the reporter construct. NIH/3T3, HaCat, CHO-K1 (Chinese hamster ovary–K1), or HEK-293 cells (7 × 104) were transiently transfected with 500 ng of a constitutive CaSR construct (pAT12, PhCMV-CaSR-pA) and 500 ng of either an optimized inducible SEAP reporter construct (pAT50, PCa6-SEAP-pA) or a mock construct [pcDNA3.1(+)] and induced with increasing Ca2+ concentrations. Increase in SEAP expression compared between 0.5 and 1.5 mM and 1.8 mM Ca2+ concentrations, P < 0.0001 at both concentrations determined using independent t test where ****P < 0.0001. (D) Cell line screening for SEAP expression. NIH/3T3, HaCat, CHO-K1, or HEK-293 cells (7 × 104) were transiently transfected with 500 ng of a constitutive CaSR construct (pAT12, PhCMV-CaSR-pA) and 500 ng of either an inducible SEAP reporter construct (pAT50, PCa6-SEAP-pA) or a mock construct [pcDNA3.1(+)] and induced with increasing Ca2+ concentrations. Increase in SEAP expression observed in HEK-293 cells between 0.5 and 2 mM Ca2+ concentrations, P = 0.0029 determined using independent t test (**P < 0.01). (E) Cell line screening for tyrosinase expression. NIH/3T3, HaCat, CHO-K1, or HEK-293 cells (7 × 104) were transiently transfected with 1 μg of a constitutive tyrosinase construct (pAT10, PhCMV-Tyr-pA). Melan-A cells were used as a positive control. Increased tyrosinase activity compared between Melan-A and CHO-K1 and HEK-293 expressing constitutive tyrosinase, P = 0.0019 and P = 0.0001, respectively, determined using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (**P < 0.01 and ****P < 0.0001). Corresponding reporter concentrations were profiled 72 hours after induction with Ca2+. A475nm, absorbance at 475 nm. All experimental data are presented as means ± SD, n ≥ 3 independent experiments.

  • Fig. 2 Characterization of the engineered HEKTattoo cells.

    (A) Time courses of tyrosinase activity of HEKTattoo cells induced with increasing Ca2+ concentrations, obtained by monitoring the rate of 3,4-dihydroxy-l-phenylalanine oxidation and the resulting melanin production for ~6 hours during culture. (B) Melanin production in HEKTattoo and photographs of the corresponding cell pellets induced with increasing Ca2+ concentrations. Increase in melanin production compared between 1.3 and 1.6 mM (P = 0.0007), 1.8 mM (P = 0.0003), and 2.0 mM (P < 0.0001), determined using independent t tests. All analyses were performed after 72 hours incubation with Ca2+. Data are shown as means ± SD, n ≥ 3 independent experiments. (C) Melanin production kinetics of HEKTattoo exposed to normocalcemia (1.3 mM), mild hypercalcemia (1.6 mM), and moderate hypercalcemia (1.8 mM) for different periods of time. Increase in melanin production by HEKTattoo exposed to Ca2+ concentrations of 1.6 and 1.8 compared to 1.3 for different time durations—24 hours: 1.3 mM versus 1.6 and 1.8 mM, P = 0.0412 and P = 0.0006, respectively; 48 hours: 1.3 mM versus 1.6 and 1.8 mM, P = 0.0001 and P = 0.0001, respectively; and 72 hours: 1.3 mM versus 1.6 and 1.8 mM, P = 0.0001 and P = 0.0001, respectively. Statistical significance of differences determined using one-way ANOVA with Dunnett’s multiple comparison test. Data are shown as means ± SD, n ≥ 3 independent experiments. (D) Stability of melanin in HEKTattoo pellets stored for up to 6 months at room temperature. (E) In vitro functionality assessment of encapsulated HEKTattoo cells in response to transient Ca2+ exposure. Microscopy images of pelleted microencapsulated HEKtattoo cells incubated under normocalcemic and hypercalcemic conditions. Scale bars, 100 μm. (F) Relative melanin concentrations in lysed microencapsulated HEKtattoo and photographs of the corrresponding microencapsulated cell pellets incubated under normocalcemic and hypercalcemic conditions. HEKTattoo cells were incubated in calcium-free media for 24 hours after encapsulation to allow Ca2+ associated with the encapsulation process to diffuse out of the capsules. Images were taken and melanin analysis was performed after incubation at normo- and hypercalcemic concentrations for 96 hours. (G) Grayscale microscopy images of porcine skin implanted with Ca+-induced (hypercalcemia) and noninduced (normocalcemia) microencapsulated HEKTattoo cells. (H) Normalized pixel intensities proportional to melanin concentration in ex vivo porcine skin implanted with HEKTattoo cells treated with hypercalcemic and normocalcemic conditions. HEKTattoo cells were preincubated in medium containing either 1.3 or 1.8 mM Ca2+ before implantation. Images were normalized to have the same mean pixel intensity of the reference standard (black sheet). Scale bars, 500 μm. Data are presented as means ± SEM, and statistical significance of differences between hypercalcemic and normocalcemic groups was calculated using unpaired t test (n = 7 injection sites) where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 3 Application of the biomedical tattoo system in nude mice.

    (A) Schematic representation of the engineered nonencapsulated HEKTattoo cells implanted into nude mice inoculated with hypercalcemic (HEK-293 with constitutive tyrosine expression, 410.4, and Colon-26 cancer cell lines) and normocalcemic cells (168 cancer cell line). (B) Photograph of a mouse from the positive control group implanted with HEK-293 cells constitutively expressing the tyrosinase construct. (C) Photograph of a negative control mouse inoculated with normocalcemic cancer (168 cell line) and implanted with HEKTattoo cells. (D) Grayscale microscopy images of the HEKTattoo implantation site in mice inoculated with 168, 410.4, and Colon-26 cell lines. Scale bars, 8.6 mM. (E) Ca2+ blood concentrations of mice in the hypercalcemic and normocalcemic groups. 168 versus 410.4 (P = 0.0001) and Colon-26 (P = 0.0001). Horizontal dashed lines represent hypercalcemia conditions (mild, 5.6 to 8 mg/dl; moderate, 8.1 to 10 mg/dl; severe, >10 mg/dl). (F) Normalized pixel intensities extracted from the skin at the implantation site of individual mice from hypercalcemic and normocalcemic groups. 168 versus 410.4 (P = 0.0001) and Colon-26 (P = 0.0001). Images were normalized to have the same mean pixel intensity of the reference standard (black sheet). (G) Melanin extracted from the implantation site of individual mice from hypercalcemic and normocalcemic groups after 24 days. 168 versus 410.4 (P = 0.0118) and Colon-26 (P = 0.0001). Data are represented as means ± SEM, and statistical significance of differences between hypercalcemic and normocalcemic groups was calculated using one-way ANOVA with Dunnett’s multiple comparison test (n ≥ 7 mice) where *P < 0.05 and ****P < 0.0001.

  • Fig. 4 Application of the biomedical tattoo system in wild-type mice using microencapsulated HEKTattoo cells.

    (A) Schematic representation of the microencapsulated engineered HEKTattoo cells implanted into wild-type mice inoculated with hypercalcemic and normocalcemic cancer cells. (B) Photograph of a mouse from the positive control group implanted with microencapsulated HEK-293 cells constitutively expressing the tyrosinase construct. (C) Photograph of a negative control mouse inoculated with normocalcemic cancer (168 cell line) and implanted with microencapsulated HEKTattoo cells, illuminated by red light. (D) Grayscale microscopy images of the pinched skin transilluminated with red light at the microencapsulated HEKTattoo implantation site of mice inoculated with 168, 410.4, and Colon-26 cell lines. Scale bars, 8.6 mm. (E) Ca2+ blood concentrations of mice in the hypercalcemic and normocalcemic groups. 168 versus 410.4 (P = 0.0001) and Colon-26 (P = 0.0001). Horizontal dashed lines represent hypercalcemia conditions (mild, 5.6 to 8 mg/dl; moderate, 8.1 to 10 mg/dl; severe, >10 mg/dl). The termination point was a tumor size of 10 mm. The last points were collected over 18 days at the terminal tumor size. (F) Normalized pixel intensities extracted from the skin images of the implantation site of hypercalcemic and normocalcemic groups after 38 days. 168 versus 410.4 (P = 0.001) and Colon-26 (P = 0.0085). Images were taken using transillumination at a wavelength of 617 to 645 nm and normalized to have the mean pixel intensity of the reference standard (gray-level reference standard). (G) In vivo melanin production by microencapsulated HEKTattoo cells extracted from the implantation site of mice in the hypercalcemic and normocalcemic groups after 38 days. 168 versus 410.4 (P = 0.0003) and Colon-26 (P = 0.0001). Data are represented as means ± SEM, and statistical significance of difference between hypercalcemic and normocalcemic groups was calculated using one-way ANOVA with Dunnett’s multiple comparison test (n ≥ 6 mice) where **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/437/eaap8562/DC1

    Table S1. Plasmids used and designed in this study.

    Table S2. Individual subject-level data.

    Movie S1. Melanin production by HEKTyr cells.

    Movie S2. HEKTattoo cells cultured in hypocalcemic medium.

    Movie S3. HEKTattoo cells cultured in normocalcemic medium.

    Movie S4. HEKTattoo cells cultured in mild hypercalcemic medium.

    Movie S5. HEKTattoo cells cultured in moderate hypercalcemic medium.

    MATLAB code

    References (6467)

  • Supplementary Material for:

    Synthetic biology-based cellular biomedical tattoo for detection of hypercalcemia associated with cancer

    Aizhan Tastanova, Marc Folcher, Marius Müller, Gieri Camenisch, Aaron Ponti, Thomas Horn, Maria S. Tikhomirova, Martin Fussenegger*

    *Corresponding author. Email: fussenegger{at}bsse.ethz.ch

    Published 18 April 2018, Sci. Transl. Med. 10, eaap8562 (2018)
    DOI: 10.1126/scitranslmed.aap8562

    This PDF file includes:

    • Table S1. Plasmids used and designed in this study.
    • Legends for movies S1 to S5
    • References (6467)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Individual subject-level data.
    • Movie S1 (.mp4 format). Melanin production by HEKTyr cells.
    • Movie S2 (.mp4 format). HEKTattoo cells cultured in hypocalcemic medium.
    • Movie S3 (.mp4 format). HEKTattoo cells cultured in normocalcemic medium.
    • Movie S4 (.mp4 format). HEKTattoo cells cultured in mild hypercalcemic medium.
    • Movie S5 (.mp4 format). HEKTattoo cells cultured in moderate hypercalcemic medium.
    • MATLAB code

    [Download Table S2]

    [Download Movies S1 to S5]

    [Download MATLAB code]

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