Highly selective behaviour of gastric adenoma after administration of EMR composition and its HCT116-based model


 When applying improved composition of solution used during endoscopic mucosal resection (EMR), we have observed unexpectedly large and quantitatively significant difference in response of adenoma vs. healthy tissue of surrounding GIT tract, namely the selective reaction enhancing the volume and differentiated colour. The in vitro experiments on model neoplasia cell line HCT116 suggest, that the robust differences in response of starving cells can be traced down principally to the tetrastarch digestion of neoplastic tissue and enhanced metabolic rate of neoplastic cells. The neoplastic tissue grows into several intestine layers so that submucosal injection of iso-oncotic tetrastarch compound leads to degradation of starch and production of oncotic molecules in submucosa transported by facilitated transport into neoplastic tissue. The colour distinction of the reporting dye is due to concentration differences of three separated compartments, further enhancing the utility of the contrasting mixture. The diffusion dynamics shall be tuneable by optimizing starch composition improving desirable pharmacokinetics.


Introduction
Neoplasms of the gastrointestinal tract are tumour-transformed tissues which, without treatment, can gradually progress into malignant tumours. The therapy of such neoplasms is usually radical, by surgical procedure-incision of detected neoplasms. The position, shape and size of the neoplasm is typically corroborated using the endoscopic method. The surgeon uses endoscopic optical probe to localize the position of such polyps along the gastrointestinal tract.
The neoplasia which is not embedded into the deeper layers of the tissue can be removed during the same endoscopic examination session by using polypectomy. By resection through the middle or deeper part of the submucosal layer, endoscopic mucosal resection (EMR) allows complete and curative resection of the diseased mucosa. For indicated early stages of progression, the EMR can be accomplished with minimal cost, morbidity, and mortality, and with the potential of improving the long-term quality of life of patients 1,2 .
Injection of suitable solution is used to separate the neoplasm from muscularis propria. If the lesion is clearly distinct visually, it usually means that there is no deep submucosal invasion. On the other hand, the "non-lifting sign" has been found to have 100% sensitivity, 99% specificity, and 83% positive predictive value for invasive carcinoma 3 .
Successful elevation of neoplasm allows the application of polypectomic loop and control of incision process. Composition of the injection solution for EMR use is not standardized 4 but efforts have been made to improve coagulant and colouring properties of such compositions. In literature, the use of coagulant as a highly concentrated salt solution with the addition of adrenalin or epinephrine is reported 5 . Alternatively, the physiological solution with addition of the derivatives of cellulose, succinyl gelatine, glycerol and fibrinogen have been formulated to slow down diffusion 4 . The physiological solution with addition of methylene blue or sodium salt of indigotindisulfonate was applied as a visual tool staining the neoplasm 5 .
The disadvantage of currently utilised solutions for diagnostics and surgical treatment of neoplasm of the GI tract is mainly short time of the elevation of the lesion (separation of adenoma) as well as only moderate visual distinction from surrounding tissue. The lifetime of such raised adenoma is determined by fast diffusion of injected solution, which leads to the disappearance of adenoma without its colour distinction. In the case where auxiliary colouring agent is used, the boundary between the adenoma and healthy tissue is dispersed due to rapid diffusion of the colouring agent into both volumes.
During our search for the optimal composition of solution, we have found the very pronounced effect when specific injection solution -composed from components approved for systemic administration and administered under valid law -was applied as submucosal injection into the neighbourhood of suspected neoplastic tissue as a part of EMR procedure. Several polyps raised above the injected tissue, lasting several minutes and forming pronounced stem allowing for comfortable application of polypectomic loop. The colouring compound formed three differently coloured volumes -injected tissue, neoplastic tissue and thin boundary between them, further helping to 6 diagnose the extent of neoplastic tissue and its level of embeddedness. The new empirically found composition clearly and repeatedly improved the EMR procedure when compared with actual clinical practice, but also when compared with literature.
In our attempt to elucidate the behaviour observed during EMR, we have performed the in vitro experiments on HCT116 (human colorectal carcinoma) cell line subjected to the conditions as present during endoscopy. In the paper we report our observations as well as the suggestion of the mechanism of action based on our limited experiments and known facts.

Materials and Methods
Preparation of EMR solution. The EMR solution we used is three-component: Adding the contrasting composition. The EMR composition was applied for two groups of cells -nonstarving and after 24 hours starving in saline solution.
After 24 hours, the non-starving cells were adherent to the density plate, while in starving cells, the free-floating cells had to be removed and media replenished by saline solution first. In both cases, the last step of the procedure consisted of replacing the saline medium by EMR composition. For starving cells about half of the volume of saline was replaced by EMR composition. Subsequently, we have prepared a set of the substance without a colour constituent. In all cases, the cells exposed to the solution were followed by 10 minutes of live video flow on a Cytation 3 Cell Imaging multimode sensor (BioTek Instruments, Inc.) and evaluated visually for cell count, cell volume and cell shape change.

Results
EMR use. Under the conditions specified above, the EMR composition was applied for 50 endoscopies with adenoma qualifying for EMR. The typical reaction to administration of contrasting composition by submucosal injection on adenoma larger than 20 mm is depicted on Figs. 1 -4. EMR composition provides colour contrasted differences between the tissues. The normal tissue is of light blue colour, the thin boundary between the adenoma and the healthy tissue is dark blue, while adenoma is not coloured. By injection of this EMR composition into submucosa layer, the adenomatous polyps increase noticeably their volume and elevate above the surface for the duration of 10-25 minutes, prolongating the time window for the resection. At the same time sharp colour differences between the healthy and the neoplastic tissue and the boundary between them can be observed. Colour distinction as well as the increased volume of the elevated polyp thus increases precision and quality of polypectomic surgery.
In vitro model. For non-starving cells, we observed no significant changes when saline solution was replaced by EMR composition. This is in sharp contrast with reaction of starving HCT116 cells. In repeated experiments, the HCT116 cells starved by 24 hours in saline solution detached completely from the density plate. On Fig. 5 and Fig. 6 we document rare case, where the group of detached cells remain partially attached, so they could be localised and recognised. In other cases, the reaction of cells was so pronounced, that the cells completely disappeared from the visual field of microscope and were not identified. For the cells on both Fig. 5 and Fig. 6 we could identify the estimated cell volume changes about 4% but for the majority of experiments, the expected volume expansion leading to the separation of cells must be higher but was not quantified.
The experiments repeated after two weeks with different HCT116 culture confirmed the same results.

Discussion
Our original attempt to improve the EMR composition led us to observation of significant differences in the response exclusively for starving cells. Starvation of in vitro cell culture was achieved by leaving the cells for 24 hours in saline only (no standard fasting medium was used). In clinical practice, the starvation is achieved by the patient abstaining from oral food and fluid intake for 24 hours (nill per os) before EMR.
The only component of EMR composition capable of triggering such reaction is HES.
The lower intestines are not involved in starch digestion so the differences must be due to starch processing capabilities of neoplastic tissue.
The administration of EMR composition into submucosa exposes the embedded part of the adenomatous polyp to the HES -chemically modified starch. HES as large macromolecule (average molecular weight 130 kDa) 9 , is expected to diffuse slowly in submucosa. Indeed, this can be seen by comparison of Fig. 2 and Fig. 3  If alpha-amylases (or their functional equivalents) are present in submucosa, the initial volume of iso-oncotic HES can be degraded, and progressively smaller hydrolysis fragments formed in submucosa. In contrast to blood plasma, even the fragments below the renal threshold (45-60 kDa) 12 remain available for further hydrolysis. As a result, continuous supply of glucose and hydroxyethyl glucose (hydroxyethylated at C2/C6 ratio 9.05:1) 9 is delivered into submucosa. The dynamic mixture of fragments including the final monosaccharide product of alpha-amylases is produced in submucosa, contributing to rise of oncotic pressure.
Without transport of monosaccharides, the oncotic pressure would manifest as volume increase of submucosa. In our observation, the dominant volume changes are observed in the volume of neoplastic tissue. The volume change is also observable in vitro at adherent 2D plaques formed by model HCT116 line. While carcinomas are known for enhanced expression of glucose transporters, adenomas must acquire the capability gradually. It seems thus reasonable, that polyps larger than 20 mm indicated for EMR possess enhanced amount of glucose transporters, particularly GLUT1 13 , 14 .
In healthy tissue, the transport of monosaccharides proceeds in direction from lumen to serosa and the transport for excessive concentration of monosaccharides is facilitated. The adenomas expose the surface present to lumen also to the submucosa, so that the direction of facilitated transport of monosaccharides is reverted.
The facilitated transport of sugars into cells is specific in the sense, that different saccharides are transported with different efficiency. Also, the hydroxyethyl glucoses are expected to transport inside the cells less efficiently than anhydrous glucose. Thus, the depletion of the pool of oncotic pressure generating saccharides in the submucosa volume follows complex kinetics, well beyond our current focus.
Nevertheless, the in vitro HCT116 model cells eagerly transport oncotic molecules, as demonstrated by violent volume changes leading to loss of cell adherence. This kinetics can be the reason that the polyp rather than submucosa increases the volume in clinical observation/application. EMR administration details. The EMR protocol which was applied corresponds to the submucosal injection of the EMR composition, where the actual composition is delivered into submucosa.
The resection starts by administration of the EMR composition below the adenomainto the healthy submucosa. The preparation of patient includes 24 hours fasting.

Conclusion
The modification of EMR composition and the application protocol lower the application barrier for EMR by improving the comfort and precision of the EMR. By The explanation we present suggest the possibility to modify the application protocol by taking advantage of different pharmacokinetics based on controlled and tuneable development of oncotic pressure. This can be probably used to develop more selective drug delivery to more specifically characterised target tissues. Fig. 1 Endoscopic findings before submucosal injection of the EMR composition. The size of the polyp is about 4,5 cm. (Time T+0 seconds immediately before submucosal injection) The same field of view as in Fig. 5 after the cells were exposed to combination saline solution and HES in a ratio 3:7. After T+30 seconds the cells detach and for most part float away from the field of view (bright field image)