Chernet BT et al. (2016), Use of genetically encoded, light-gated ion tra...

XB-ART-51992

Oncotarget 2016 Apr 12;715:19575-88. doi: 10.18632/oncotarget.8036.

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Use of genetically encoded, light-gated ion translocators to control tumorigenesis.

Chernet BT , Adams DS , Lobikin M , Levin M .

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It has long been known that the resting potential of tumor cells is depolarized relative to their normal counterparts. More recent work has provided evidence that resting potential is not just a readout of cell state: it regulates cell behavior as well. Thus, the ability to control resting potential in vivo would provide a powerful new tool for the study and treatment of tumors, a tool capable of revealing living-state physiological information impossible to obtain using molecular tools applied to isolated cell components. Here we describe the first use of optogenetics to manipulate ion-flux mediated regulation of membrane potential specifically to prevent and cause regression of oncogene-induced tumors. Injection of mutant-KRAS mRNA induces tumor-like structures with many documented similarities to tumors, in Xenopus tadpoles. We show that expression and activation of either ChR2D156A, a blue-light activated cation channel, or Arch, a green-light activated proton pump, both of which hyperpolarize cells, significantly lowers the incidence of KRAS tumor formation. Excitingly, we also demonstrate that activation of co-expressed light-activated ion translocators after tumor formation significantly increases the frequency with which the tumors regress in a process called normalization. These data demonstrate an optogenetic approach to dissect the biophysics of cancer. Moreover, they provide proof-of-principle for a novel class of interventions, directed at regulating cell state by targeting physiological regulators that can over-ride the presence of mutations.

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Species referenced: Xenopus laevis
Genes referenced: actc1 cdh15 kras myod1 pnma2 slc5a5

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	Figure 1. Optogenetic modulation of Vmem to control ITLSs is achieved using a KRASG12D oncogene and light-sensitive ion channels in Xenopus laevis embryos(A) ITLSs were generated by injecting KRASG12D mRNA into a single blastomere of 16 cell stage embryos. Injected embryos were raised in 0.1 Ã MMR before they were scored for the presence of ITLSs and imaged using bright field microscopy between stages 28 and 35. (B) Schematic of optogenetic Vmem modulation using a Channelrhodopsin-2 mutant (ChR2D156A) channel: ChR2D156A mRNA was injected into 1 cell of a 2-cell embryo, allowing the uninjected side to serve as an internal control. Embryos were raised to stage 18 in 0.1XMMR. (C) At stage 18, embryos were soaked in 1.9 Î¼M DiBAC4(3) solution in 0.1 Ã MMR, and imaged using a DiBAC4 (3) filter set (470/20; BS 485; EM 517/23). The un-injected left half of the embryo was highly fluorescent, indicating relative depolarization compared to the right half of the embryo, which is expressing ChR2D156A. Scale bar = 150 Î¼m.
	Figure 2. Set up used to deliver spatio-temporally precise light stimulation of optogenetic ion-translocators expressed in Xenopus embryo ITLSsWe customized a Nikon AZ100 dissection scope for in vivo optogenetics [87] by replacing the epifluorescence illumination source and light guide with a Spectra4 LED illuminator connected to the scope via fiber optic cable (the black light guide that passes in front of the vacuum source). The light is passed through a pinhole to set spot size diameter (located behind the oculars, not visible in this image), then enters the scope and goes through an 80/20 splitter that allows the user to view the specimen on the monitor even while the LED is on. Finally, the light passes through the 5 Ã objective lens which further reduces spot diameter and aims the spot at the sample. An automated Ludl MAC6000 XY stage (that can also be manipulated manually by a joystick) allows multiple embryos to be exposed repeatedly to the activating wavelength of light. Up to thirty embryos are loaded into a slide-mounted PDMS âchipâ designed to use microfluidics to hold embryos in place [100]; the chip is held to the slide by a vacuum and 0.1 Ã MMR is circulated by a peristaltic pump. The optogenetics components and the microscope are all controlled by NIS Elements.
	Figure 3. Expression of optogenetic channels Arch and ChR2D156A prior to ITLS appearance suppresses the formation of KRASG12D ITLSs(A) Schematic of experimental design for ITLS suppression: mRNAs for KRASG12D and ChR2D156A or Arch were co-injected into a single blastomere of 16-cell stage embryos; between 4 and 28 hours post injection (a total exposure of 24 hours), injected embryos were exposed to 580 nm wavelength of light, 1 mw/mm2 irradiance (stimulating Arch) or 450 nm wavelength of light with 2.4 mw/mm2 irradiance (stimulating ChR2D156A). Arch and ChR2D156A experiments were done separately to test the hypothesis that ITLS suppression is due to changes in membrane voltage as opposed to channel or ion specific properties. Light-stimulated embryos were raised to stage 35 and scored for ITLSs to assess the efficacy of hyperpolarizing optogenetic channels as ITLS suppressing reagents. (B) The two different hyperpolarizing translocators â based on active pumping of H+ (Arch) and passive diffusion of positive, monovalent cations (ChR2D156A) â both resulted in suppression of ITLSs, demonstrating that this effect is likely due to a change in Vmem and is not tied to one channel protein. 32% and 31% fewer embryos with ITLS were observed among Arch and ChR2D156A injected embryos, respectively (Arch: c2 = 7.1, p = 0.007; ChR2D156A: Ï2 = 6.8, p = 0.009. Ï2 values are for comparisons of ITLS incidence in light stimulated versus un-stimulated embryos).
	Figure 4. After ITLS formation, activation of ChR2D156A normalizes KRASG12D ITLSs(A) Schematic of experimental design for ITLS normalization: ChR2D156A expressing embryos (stages 28â35) with KRASG12D ITLSs were subjected to 450 nm wavelength of light with irradiance of 0.5 mw/mm2. Light-stimulated embryos were raised to stage 45â47 and scored for ITLSs to assess the ability of optogenetic Vmem modulation to normalize ITLSs. (B) Light activation of ChR2D156A in ITLSs (blue bar) resulted in 31% more embryos with normalized ITLSs compared to their un-stimulated counterparts (red bar) (Ï2 = 8.6, *p = 0.003). By comparison, treatment with the highly-selective MEK 1 inhibitor (Selumetinib), a potent PI3K inhibitor (Pictillisib), or a potent inhibitor of oncogenic B-RAF kinase activity (Vemurafenib), resulted in the same KRASG12D tumor incidence as in the unstimulated control.
	Figure 5. Muscle specific expression of GFP3 controlled by the cardiac actin (Car) promoter is present in KRASG12D ITLSTadpoles injected with Tol2-CarPr-GFP3 [101] at the 2-cell stage display normal muscle specific localization of GFP3 within the somites (A, Aâ², Aâ showing transmitted light, GFP fluorescence, and both, respectively). In contrast, Tol2-CarPr-GFP3 tadpoles also injected with KRASG12D reveal a strong GFP3 signal in ITLS's, confirming the presence of ectopic muscle in the KRAS-induced tumor-like structures.
	Figure 6. Light activation of ChR2D156A results in downregulated expression of ectopic muscle markersHuman tumors associated with KRAS mutations express the muscle markers myod1 and cdh15. Using qPCR (see Materials and Methods for details), we found that expression of myod1 and cdh15 was likewise up-regulated in tadpoles with KRASG12D-induced ITLSs. Moreover, activation of ChR2D156A in these tadpoles significantly lowered myod1 and cdh15 expressions, down to baseline levels (Ï2 test compared to controls, *p < 0.01).

References [+] :

Adams, Measuring resting membrane potential using the fluorescent voltage reporters DiBAC4(3) and CC2-DMPE. 2012, Pubmed, Xenbase