**7. The amino-acids treatment in the Electron-Beam Plasma**

294 Practical Applications in Biomedical Engineering

calculated as the (*mdr/ ms*)100% ratio.

Fourier analysis was performed.

mesuarment was 10%.

experiments.

temperature - 30оС; UV-detector with the wavelength 280 nm.

immunoelectrophoresis and PAGE-electrophoresis as well [12].

*Solubility measurements.* 100±0,1 mg of the preliminary dried sample (*ms*) were placed into a tube and 1,5 ml of distilled water were added to the sample. The resulting mixture was incubated for 24 h at room temperature under periodic mixing. After the incubation the mixture was centrifuged for 5 min and 1 ml of centrifugate was taken and dried. The mass of the dry residue (*mdr*) was measured with an accuracy ±0,1 mg. The sample solubility was

*Molecular mass characterization.* To characterize the molecular masses of the EBP-treatment products the exclusion chromatography was applied. The chromatograph *Staier* (Russia) and the chromatographic column *Phenomenex* BioSep-Sec-S-3000 (USA) with the efficiency of 30000 theoretical plates were used. The analysis conditions were as follows: the elutriating agent – 0,1 M phosphate buffer (pH 6,86) containing 0,05% NaN3; the elution rate - 1 ml/min;

The effects of the EBP-treatment on proteins molecular mass and structure were detected also by means of the UV- and IR-spectroscopy, ion-exchange chromatography,

*UV-spectroscopy*. The measurements were performed with the spectrometer *Shimadzu UV-*

*IR-spectroscopy*. The measurements were performed with the IR-spectrometer *Portmann Instruments AG* (Switzerland) equipped with the ZnSe crystal. The IR-spectra were registered within wave numbers *ν* = 500-3600 cm-1. To improve the spectral resolution

*Ion-exachange chromatography.* The ion-exchange chromatography (with the preliminary acid hydrolysis of the protein) was performed to reveal the changes in the amino acid composition of proteins due to the EBP-modification. The analyzer *AAA-339 M* (Hungary) was used. To quantitatively analyze the sulfur-containing amino acids (cystine and methionine) the biomaterial was treated with the performic acid before the hydrolysis procedure. Totally 17 basic amino acids contents were measured. The accuracy of the

*Immunoelectrophoresis.* The electrophoresis was performed in the 1,4% agar gel [13]. The commercial specific antiserum to human fibrinogen was used to characterize and compare

*Biological activity of the EBP-produced low molecular weight chitosans (LMCW).* The inhibition of the bacteria growth *in vitro* was measured to quantitatively characterize the bioactivity of LMCW obtained by the plasma treatment, gram-positive (*S. aureus*), gram-negative (*E. coli, Ps. aeruginosa*) microorganisms and yeast-like fungi (*C. albicans*) being used in these

antigenic structure of the FM before and after plasmachemical treatment.

PAGE-*electrophoresis* was performed according to Laemmli U.K. [14].

*3600* (Japan). The IR-spectra were registered within wavelength λ = 226-418 nm.

Some natural amino acids with artificially inserted pirozolidine cycles into their structures were used as original substances and the products of their modification in the EBP of helium and water vapor were tested as inhibitors of the human platelet aggregation. Preliminary analysis showed the substances of this class to be promising as active agents for medical therapy of acute coronary events, and cardiovascular diseases that remain the leading cause of mortality. Their advantages are due to selectivity of the pharmacology action and limited side effects.

The powder samples (≈ 50 mg in mass) of the original derivative of alanine were treated in the EBP of water vapor at pressure *Pm* ≈ 9 Torr for variable time duration *τ* = 45-300 s. The typical EB power was *Nb* ≈ 0,1 kW, the sample temperature *Ts* under the treatment could be varied within the range 30-110 °C.

The untreated compound was not dissolvable in distilled water at room temperature and the water heating up to 90 °C followed by cooling to 25 °C was required to carry out the control experiments and to study its effect on human platelet aggregation *in vitro*. The treated substance became partially water-soluble at room temperature and the solution at maximum concentration was added to the platelet suspension to measure the aggregation degree.

The untreated derivative decreased human platelet aggregation only to 46±2% with respect to control (56±2%). The water-soluble products of plasma treatment reduced the aggregation degree up to ≈ 30 %, i.e. being treated by the EBP for 5 min the studied substance reduced the platelet aggregation activity by approximately 45 % (Table 1).

**Table 1.** The effect of the plasma modification in the EBP of water vapor on the anti-aggregation activity of the tested alanine derivative (*in vitro*): the aggregation degree as a function of the treatment duration and temperature of the substance *T*s under the treatment procedure

The effect of the treatment duration on their anti-aggregation activity increased as the treatment prolonged, the anti-aggregation activity rising sharply at 90 < *τ* < 180 s. At shorter durations *τ* < *τ0* the plasma did not modify the original substance and the longer treatment *τ*

> τ0 resulted in insignificant additional effect. Moderate sample heating amplified the treatment effect slightly, i.e. plasmachemical processes are responsible for the modification.

Bio-Medical Applications of the Electron-Beam Plasma 297

confirms the partial destruction of the native protein molecule and low molecular

3. The spectral bands ν = 1163 cm-1 and ν = 1471 cm-1 which characterize the oxidation of

These facts confirm that the EBR-treatment of the FM for = 5 min has caused the partial destruction of the peptide –CO-NH-bonds in the primary FM structure and the oxidation of

All changes were more significant in the case of the water vapor EBP, which could result

disulfide bounds are most intensive in the IR-spectra of EPB-treated FM.

from the higher chemical activity of the water plasmolysis products (e.g. OH•).

**Figure 6.** The immunoelectrophoresis of FM before and after the EBP-treatment

chromatograms of the FM modified by the EBP (Figure 8).

The plasma treatment reduced the amount of amino acids forming the primary protein structure (the content of 17 amino acids was studied). The percentages of lysine, threonine, glutamic acid, cystine, tyrosine and phenylalanine were found to be reduced significantly (down to 2 times with respect to the native FM, Table 2). The reduction of aspartic acid, serine, glycine, valine, methionine, leucine and isoleucine was not so sharp (only 1,3-1,5 times with respect to the native FM). The intensive peak of free NH3 was detected in the chromatogram of the modified products whereas it was not found in the chromatogram of

To characterize the molecular masses of the EBP-treatment products the exclusion chromatography was applied. The peaks corresponding to 6 individual peptides with the elution times 12,30; 12,55; 13,17; 13,70; and 13,94 min were observed in the exclusion

The water-soluble products of the FM appropriately treated by the EBP of both helium and water vapor were found to decrease the platelet aggregation down to 33-35 % *in vitro* at final concentrations 1×10-5-1 mg/ml, treatment in the water vapor EBP being more effective

the disulfide bonds responsible for the tertiary peptides structure.

weight peptides formation.

the original peptide.

### **8. The proteins treatment in the Electron-Beam Plasma**

Originally water-indissoluble native FM was found to become soluble at room temperature without bunching. Figure 5 presents the UV-spectra of absorption for original and treated fibrin-monomer. The spectral curves of the modified products differ radically from the curve representing the original substance. This supports the hypothesis of the changes in the physical-chemical properties due to the plasmachemical treatment in the EBP.

**Figure 5.** UV-spectra of the light absorption of fibrin-monomer solutions

Radical changes in the FM structure after the EBP-treatment were detected by means of immunoelectrophoresis. The EBP-modified products did not exhibit the specific antigenic properties of original FM and did not react with specific antibodies, while the native substance gave specific precipitation line (Figure 6).

To reveal the changes in the primary and secondary structure of the EBP-treated FM the IRspectroscopy was used. The IR-spectra analysis showed (Figure 7 a, b):

confirms the partial destruction of the native protein molecule and low molecular weight peptides formation.

3. The spectral bands ν = 1163 cm-1 and ν = 1471 cm-1 which characterize the oxidation of disulfide bounds are most intensive in the IR-spectra of EPB-treated FM.

296 Practical Applications in Biomedical Engineering

> τ0 resulted in insignificant additional effect. Moderate sample heating amplified the treatment effect slightly, i.e. plasmachemical processes are responsible for the modification.

Originally water-indissoluble native FM was found to become soluble at room temperature without bunching. Figure 5 presents the UV-spectra of absorption for original and treated fibrin-monomer. The spectral curves of the modified products differ radically from the curve representing the original substance. This supports the hypothesis of the changes in the

**8. The proteins treatment in the Electron-Beam Plasma**

**Figure 5.** UV-spectra of the light absorption of fibrin-monomer solutions

spectroscopy was used. The IR-spectra analysis showed (Figure 7 a, b):

destruction of peptide –CO-NH-bonds in the primary FM structure.

substance gave specific precipitation line (Figure 6).

Radical changes in the FM structure after the EBP-treatment were detected by means of immunoelectrophoresis. The EBP-modified products did not exhibit the specific antigenic properties of original FM and did not react with specific antibodies, while the native

To reveal the changes in the primary and secondary structure of the EBP-treated FM the IR-

1. The maximum of the absorbance in the band complex ν = 1711-1714 cm-1 (complex of amide II bands) displaced from 1711 cm-1 (native FM) to 1712 cm-1 and 1712 cm-1 (FM treated in the EBP of helium and water vapor, respectively). This indicates the partial

2. The spectral bands ν = 3354 cm-1 and ν = 3475 cm-1 characterize the valence oscillations of –N-H-bonds. The expansion of this band in the spectra of the EBP-modified FM

physical-chemical properties due to the plasmachemical treatment in the EBP.

These facts confirm that the EBR-treatment of the FM for = 5 min has caused the partial destruction of the peptide –CO-NH-bonds in the primary FM structure and the oxidation of the disulfide bonds responsible for the tertiary peptides structure.

All changes were more significant in the case of the water vapor EBP, which could result from the higher chemical activity of the water plasmolysis products (e.g. OH•).

**Figure 6.** The immunoelectrophoresis of FM before and after the EBP-treatment

The plasma treatment reduced the amount of amino acids forming the primary protein structure (the content of 17 amino acids was studied). The percentages of lysine, threonine, glutamic acid, cystine, tyrosine and phenylalanine were found to be reduced significantly (down to 2 times with respect to the native FM, Table 2). The reduction of aspartic acid, serine, glycine, valine, methionine, leucine and isoleucine was not so sharp (only 1,3-1,5 times with respect to the native FM). The intensive peak of free NH3 was detected in the chromatogram of the modified products whereas it was not found in the chromatogram of the original peptide.

To characterize the molecular masses of the EBP-treatment products the exclusion chromatography was applied. The peaks corresponding to 6 individual peptides with the elution times 12,30; 12,55; 13,17; 13,70; and 13,94 min were observed in the exclusion chromatograms of the FM modified by the EBP (Figure 8).

The water-soluble products of the FM appropriately treated by the EBP of both helium and water vapor were found to decrease the platelet aggregation down to 33-35 % *in vitro* at final concentrations 1×10-5-1 mg/ml, treatment in the water vapor EBP being more effective

than that in helium (Figure 9). The effect of the treatment on the FM anti-aggregation activity increased sharply with the time within the range 90 < < 180 s. The longer treatment resulted in a negligible additional effect. The peak corresponding to the elution time 12,3 min (molecular weight 650 Da) was observed at the exclusion chromatograms of the FM modified in the EBP of helium and EBP of water vapor at a moderate irradiation dose whereas the FM treated EBP of water vapor at lower or higher irradiation doses did not produce the peak (Figure 8). This peptide is likely to be responsible for the inhibiting of the platelet aggregation.

Bio-Medical Applications of the Electron-Beam Plasma 299

FM, treated in helium EPB

Amino acid Amino acid content, %

Lysine 0,26 0,13 0,13 Threonine 0,24 0,15 0,18 Glutamic acid 0,53 0,36 0,40 Cystine 0,068 0,039 0,051 Tyrosine 0,1 0,09 0,09 Phenylalanine 0,16 0,09 0,10 Aspartic acid 0,64 0,49 0,53 Serine 0,29 0,22 0,21 Glycine 0,24 0,22 0,22 Valine 0,15 0,12 0,13 Methionine 0,15 0,11 0,12 Leucine 0,24 0,17 0,19 Isoleucine 0,14 0,10 0,12

**Table 2.** The amino acid content of FM before and after EBP-treatment

**Figure 8.** The chromatograms of the FM treated in the EBP of helium (curve 1), water vapor at

moderate irradiation dose ( curve 2) and at low irradiation dose (curve 3)

Native FM FM, treated in water

vapor EPB

**Figure 7.** a. IR-spectra of the native and EBP-treated fibrin-monomer (τ = 5 min): native FM – blue line, FM treated in the EBP of helium – red line, FM treated in the EBP of water vapor – green line; b.IRspectra of the native and EBP-treated fibrin-monomer (τ = 5 min): native FM – red line, FM treated in the EBP of helium – blue line, FM treated in the EBP of water vapor – green line

**Table 2.** The amino acid content of FM before and after EBP-treatment

298 Practical Applications in Biomedical Engineering

platelet aggregation.

than that in helium (Figure 9). The effect of the treatment on the FM anti-aggregation activity increased sharply with the time within the range 90 < < 180 s. The longer treatment resulted in a negligible additional effect. The peak corresponding to the elution time 12,3 min (molecular weight 650 Da) was observed at the exclusion chromatograms of the FM modified in the EBP of helium and EBP of water vapor at a moderate irradiation dose whereas the FM treated EBP of water vapor at lower or higher irradiation doses did not produce the peak (Figure 8). This peptide is likely to be responsible for the inhibiting of the

**Figure 7.** a. IR-spectra of the native and EBP-treated fibrin-monomer (τ = 5 min): native FM – blue line, FM treated in the EBP of helium – red line, FM treated in the EBP of water vapor – green line; b.IRspectra of the native and EBP-treated fibrin-monomer (τ = 5 min): native FM – red line, FM treated in

the EBP of helium – blue line, FM treated in the EBP of water vapor – green line

**Figure 8.** The chromatograms of the FM treated in the EBP of helium (curve 1), water vapor at moderate irradiation dose ( curve 2) and at low irradiation dose (curve 3)

Bio-Medical Applications of the Electron-Beam Plasma 301

Whereas the products of the EBP-induced polymerization and very small concentration of low molecular peptides are detected the most significant peak (>2400 mV) corresponds to protein with molecular mass 66 kDa i.e. to the unmodified BSA. This confirms that though the EBP-treatment of the BSA causes some changes in its structure they are not as pronounced as in the case of the fibrin-monomer treatment. Therefore the modification effect of the plasma treatment depends on the size and structure of the original protein

**Figure 10.** PAGE-electrophoresis of four BSA samples: 1 – control BSA; 2, 3 and 4 – BSA treated in the

**1 2 3 4**

**Figure 11.** The exclusion chromatogram of BSA powder treated in EBP of oxygen (τ = 10 min)

On the other hand the small concentration of low molecular weight peptides is probably due to a small depth (a few micrometers) of a powder particle involved in the plasmachemical treatment. To enhance the homogeneity of the EBP-modification, the samples of globular protein were treated in the form of thin films. Such treatment arrangement can considerably

EBP of water vapor for 2,5, 5 and 10 min, respectively

molecule.

**Figure 9.** The reduction of the ADP-induced platelet aggregation by products of the FM modification in the EBP of water vapor and helium

The powder of globular protein BSA was treated in the EBP as well. The solubility of the native BSA was 100%. Contrary to the EPB-treated FM the products of the EBP-modification became partially indissoluble in distilled water and the solubility decrease could be observed by the unaided eye. The water solution of the treated BSA contained the of undissolved protein particles, the solution of native BSA was absolutely clear. The latter indicates that the BSA polymerization has occurred due to the EBP-treatment.

No differences in either BSA structure were found in the IR-spectrograms, PAGEs (Figure 10), changes in its amino acid composition were not observed also. Since the molecular mass of the BSA is significantly less than that of the fibrin-monomer the BSA may be more resistant to the EBP treatment and no changes in the amino acids content and their structure in the EBP-modified BSA occur. On the other hand, minor quantities of the low molecular products which could be formed due to the EBP-treatment of the BSA are undetectable by the relatively low-sensitive PAGE technique.

The exclusion chromatography, that has higher sensitivity, was used to detect the products of the BSA treatment. The peaks corresponding to the individual peptides with molecular weights >800 kDa, 350 kDa, 150 kDa, 66 kDa and less (elution times 5,6; 7,2; 8,0; 8,7 min and more than 8,7 min, respectively) were observed in the exclusion chromatograms of the BSA modified by the EBP of both oxygen and water vapor (Figure 11).The treatment of the BSA for longer time (up to 20 min) did not result in the additional changes of its properties and composition.

Whereas the products of the EBP-induced polymerization and very small concentration of low molecular peptides are detected the most significant peak (>2400 mV) corresponds to protein with molecular mass 66 kDa i.e. to the unmodified BSA. This confirms that though the EBP-treatment of the BSA causes some changes in its structure they are not as pronounced as in the case of the fibrin-monomer treatment. Therefore the modification effect of the plasma treatment depends on the size and structure of the original protein molecule.

300 Practical Applications in Biomedical Engineering

the EBP of water vapor and helium

the relatively low-sensitive PAGE technique.

composition.

**Figure 9.** The reduction of the ADP-induced platelet aggregation by products of the FM modification in

The powder of globular protein BSA was treated in the EBP as well. The solubility of the native BSA was 100%. Contrary to the EPB-treated FM the products of the EBP-modification became partially indissoluble in distilled water and the solubility decrease could be observed by the unaided eye. The water solution of the treated BSA contained the of undissolved protein particles, the solution of native BSA was absolutely clear. The latter

No differences in either BSA structure were found in the IR-spectrograms, PAGEs (Figure 10), changes in its amino acid composition were not observed also. Since the molecular mass of the BSA is significantly less than that of the fibrin-monomer the BSA may be more resistant to the EBP treatment and no changes in the amino acids content and their structure in the EBP-modified BSA occur. On the other hand, minor quantities of the low molecular products which could be formed due to the EBP-treatment of the BSA are undetectable by

The exclusion chromatography, that has higher sensitivity, was used to detect the products of the BSA treatment. The peaks corresponding to the individual peptides with molecular weights >800 kDa, 350 kDa, 150 kDa, 66 kDa and less (elution times 5,6; 7,2; 8,0; 8,7 min and more than 8,7 min, respectively) were observed in the exclusion chromatograms of the BSA modified by the EBP of both oxygen and water vapor (Figure 11).The treatment of the BSA for longer time (up to 20 min) did not result in the additional changes of its properties and

indicates that the BSA polymerization has occurred due to the EBP-treatment.

**Figure 10.** PAGE-electrophoresis of four BSA samples: 1 – control BSA; 2, 3 and 4 – BSA treated in the EBP of water vapor for 2,5, 5 and 10 min, respectively

**Figure 11.** The exclusion chromatogram of BSA powder treated in EBP of oxygen (τ = 10 min)

On the other hand the small concentration of low molecular weight peptides is probably due to a small depth (a few micrometers) of a powder particle involved in the plasmachemical treatment. To enhance the homogeneity of the EBP-modification, the samples of globular protein were treated in the form of thin films. Such treatment arrangement can considerably

increase the yield of low molecular weight products with the comparison to the powdered samples.

Bio-Medical Applications of the Electron-Beam Plasma 303

BSA, treated in oxygen EPB τ = 10 min

BSA, treated in oxygen EPB τ = 5 min

Amino acid Amino acid content, % Native BSA BSA, treated in

Lysine 10,25 3,68 2,73 2,45 Threonine 5,02 2,65 2,63 2,27 Glutamic acid 16,78 5,78 6,64 5,90 Cystine 4,73 1,78 2,07 1,75 Tyrosine 4,30 1,70 1,55 1,79 Phenylalanine 5,01 3,43 3,37 2,90 Aspartic acid 9,17 3,86 5,12 5,14 Serine 3,75 2,03 2,11 1,89 Glycine 1,45 2,13 2,48 2,54 Valine 4,45 3,70 3,25 3,51 Methionine 2,02 0,76 0,89 0,63 Leucine 9,61 6,55 6,05 5,47 Isoleucine 1,85 1,34 3,37 2,90

water vapor EPB τ = 5 min

**Table 3.** The amino acid content of BSA before and after EBP-treatment (BSA was treared as thing film)

1. Our experiments show that the powders of various proteins can be effectively modified in the EBP and that the plasmachemical processes are responsible for the modification. With the use of special procedures of sample preparation for the EBPtreatment, it is possible not only to increase the yield of plasma modification products but also to effectively control their composition. It was found that unlike powdered samples, the materials in the form of a freeze dried film of ~1 μm thickness can be homogeneously treated to an extent sufficient for the practical use of the compounds

2. Plasmachemical reactions in thin films occur throughout the entire volume of the sample, leading to the formation of final low molecular mass products. The powdered samples are characterized by a thin surface layer (of at most 10 μm in thickness) in which the effective modification of the protein takes place. The protein remains intact in deep layers of such samples, and polymerization of protein molecules is likely to occur in intermediate layers. Our results are consisted with the data obtained in some other studies [17, 18]. The destruction of BSA thing films was also observed in the experiments using the atmospheric pressure glow discharge. Nevertheless the effective BSA degradation in the atmospheric pressure glow discharge was observed when the very small amounts of the protein sample (~20 μl) were treated [18]. Thus, the EBPtreatment seems to be more powerful technique for the protein degradation and low

molecular peptides formation with respect to the gas-discharge plasmas.

The following factors influence the biomaterial placed into the plasma cloud:

**9. Discussions and conclusions**

produced.

The PAGE of the BSA samples modified as films in EBP revealed a dramatic decrease in the intensity of the albumin band and the formation of a number of low molecular peptides, of which most have a molecular mass below 14 kDa. The exclusion chromatograms also displayed numerous peaks corresponding to low molecular mass products with elution times of 11,8; 15,1; and 17 min (Figure 12). The concentration of the low molecular mass compounds depended on the plasma treatment time and the nature of the plasma gas: as the treatment time increased, their concentration grew, and BSA degradation being more significant in the water vapor EBP. A decrease in the content of almost all amino acids was observed. The most significant decrease (by 2–3,5 times with respect to untreated BSA) was in the case of lysine, aspartic and glutamic acids, tyrosine, and cystine (Table 3).

**Figure 12.** The exclusion chromatogram of BSA thing film treated in EBP of oxygen (τ = 10 min)

**Table 3.** The amino acid content of BSA before and after EBP-treatment (BSA was treared as thing film)


**6.1 Advancing ctDNA detection accuracy**

*Advances in the Molecular Understanding of Colorectal Cancer*

wild type [66].

primary resistance [84].

mutant *KRAS* were associated with 0% disease control rate [85]. More recently, a large retrospective exploratory analysis used BEAMing technology to identify *KRAS*, *PIK3CA* and *BRAF* mutations in the plasma ctDNA of 503 patients who enrolled in the CORRECT trial of regorafenib, a multi-kinase inhibitor in refractory mCRC [66]. Tumour-associated *KRAS* mutations were readily detected with BEAMing of plasma DNA and were identified in 48% of patients who had previously received anti-EGFR therapy and whose archival tumour tissues were KRAS

Beyond *KRAS* mutations and amplifications, acquired genetic aberrations in other genes have been found to potentially lead to anti-EGFR therapy resistance, albeit in smaller subsets of patients. For example, emerging *EGFR* extracellular domain (ECD) mutations which lead to impaired antibody binding were found to be a resistance mechanism to anti-EGFR therapy in approximately 20% of patients. Interestingly, these mutations tend to arise later than *RAS* mutations during therapy, and patients with *EGFR* ECD mutations had greater and more durable response to anti-EGFR therapy than patients with *RAS* mutations [86]. Interestingly, a phase I trial of a third generation EGFR-targeting agent that binds multiple regions of the EGFR ECD demonstrated efficacy in patients with *EGFR* ECD mutations and acquired resistance to prior EGFR blockade [87]. Other genomic alterations linked to acquired resistance to EGFR blockade include *MET and ERBB2* amplifications [88, 89] and mutations in *NRAS*, *BRAF* and *PIK3CA* [6]. *ERBB2* amplification was found in the plasma in four out of eight *RAS* wild type patients who derived no clinical benefit from anti-EGFR treatment, suggesting that it may also be a source of

Another innovative study provided proof-of-principle that parallel analysis of patient-derived xenografts and ctDNA allowed the identification of resistance mechanisms to a pan-tropomyosin-related kinase (TRK) inhibitor in mCRC, with validation in preclinical models [90]. In interpreting these translational findings, it is important to note that typically, multiple complex molecular abnormalities emerge rather than a singular clone and an overlap exists between abnormalities

CtDNA genotyping has now paved the way for prospective clinical trials which aim to evaluate a range of targeted agents in mCRC and their resistance mechanisms. However, significant knowledge gaps exist in the field, including lack of standardisation of ctDNA techniques, clinical relevance of minority clones detected (for example, no threshold for *KRAS* allele frequency has been established to predict anti-EGFR therapy resistance) and it remains to be proven that changing treatment strategy according to ctDNA findings improves patient outcomes [6]. Challenges notwithstanding, it is foreseeable that in the near future, ctDNA genotyping may be used longitudinally to (i) identify *RAS* wild type patients with mCRC who may be suitable for anti-EGFR antibodies, (ii) dynamically assess treatment response, (iii) identify patients who are developing acquired resistance, (iv) delineate resistance mechanisms to therapy, and (v) discover new druggable

Despite growing enthusiasm, ctDNA in CRC remains largely unavailable for clinical application outside of the trial setting. Recently, there has been a surge of research to further investigate the utility of more sensitive and accurate technologies for ctDNA detection and analysis, and to further elucidate its clinical imple-

mentation and significance in the various settings of CRC management.

associated with primary and secondary resistance [6].

**64**

targets.

**6. Future directions**

Improved sensitivity techniques with the use of targeted-sequencing methods have been developed by several groups [9, 91]. For example, Lanman et al. validated the analytical and clinical use of a novel, ultra-high specific, digital sequencing technique (Guardant360) consisting of 54 clinically actionable cancer genes [91]. In 165 consecutively matched plasma and tumour tissue samples from patients with advanced cancer, this study demonstrated significantly improved sensitivity for Guardant360 in the plasma-derived cfDNA compared to that of tumour tissue. It also demonstrated the clinical success rate of the assay in 1000 consecutive plasma samples in the clinic (assay failure rate of 0.02%) due to its ability to eliminate false positives [91].

Other investigators have combined the use of DNA fragment sequencing by using molecular barcodes with relevant bioinformatics filtering steps to enhance sensitivity and specificity [30, 69, 92, 93]. In a study using cfDNA from mCRC patients, Mansukhani et al. showed that false positive mutation calls could be reduced by 98.6% when incorporating novel molecular barcodes for error correction and by applying custom solution hybrid capture enrichment [93].


*4.19. Statistical Analysis*

Statistical analysis was performed applying GraphPad Prism software (GraphPad Inc., San Diego, CA, USA, version 8.2.1). Variable distribution was tested for normality using the Kolmogorov-Smirnov test. Respectively, Gaussian distributed, and non-Gaussian distributed patient samples were statistical analyzed using two-tailed paired student's *t*-test or Wilcoxon matched-pairs signed-ranks test. In vitro experiments were analyzed using one-way ANOVA. Cell culture experiments were performed at least three times (independent experiments using technical replicates). Patient samples were used in experiments upon availability. P values were considered significant at \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

#### **5. Conclusions**

This study provides new insights of a significantly altered iron metabolism in renal cell carcinoma. Most of the studied iron-regulated genes are associated with tumor grade and tumor pT-stage. Moreover, our results suggest that tumor-associated macrophages adopt a pro-tumorigenic iron-releasing phenotype through increased expression of FPN. These tumor-associated macrophages are then able to fuel the increased iron demands of tumor cells by secreting iron in the tumor microenvironment.

EC1, a novel iron chelator, specifically scavenges iron in the extracellular space and was able to reverse pro-tumorigenic effects of macrophage-conditioned media on proliferation and migration of cancer cells, including primary patient-derived renal cancer cells. These results might pave the way towards further in vivo studies addressing the possibility to interfere with iron availability in the tumor microenvironment by targeted chelation strategies.

#### **6. Patents**

Issued patent: Tomat E.; Chang, T. M. "Redox-Directed Chelators Targeting Intracellular Metal Ions" U.S. Patent No. 9,486,423, November 8th, 2016.

Pending patent application: Tomat, E.; Chang, T.; Akam, E. A. "Redox-activated Pro-chelators" U.S. Patent Appl. No.: 16/200,286, November 26th, 2018.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6694/12/3/530/s1, Figure S1: Primary human renal tissue verification, Figure S2: Perl's staining of histopathological renal cell carcinoma subtypes. Figure S3: Characterization of the extracellular iron chelator EC1.

**Author Contributions:** Conceptualization: M.J.; Methodology: M.S., C.R., C.M., E.T., E.A.A., M.J.; Software: M.S.; Validation: M.S., C.R., C.M., J.K.M., M.J.; Formal analysis: M.S., C.R., A.U., C.M., E.T., E.A.A., M.J.; Investigation: M.S., C.R., A.U., C.M., J.K.M., E.T., E.A.A., P.B., F.C.R., M.J.; Resources: A.U., P.B., F.R., B.B., M.J.; Data curation: M.J.; Writing—original draft preparation: M.S., E.T., B.B., M.J.; Writing—review and editing, M.S., E.T., B.B., M.J.; Visualization: M.S., M.J.; Supervision: M.J.; Project administration: M.J.; Funding acquisition: E.T., B.B., M.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** MS—PhD scholarship from the Faculty of Medicine, Goethe-University Frankfurt; CR—PhD scholarship from EKFS (Else Kröner-Fresenius-Graduiertenkolleg); BB—Else Kröner-Fresenius Foundation (EKFS), Research Training Group Translational Research Innovation–Pharma (TRIP); MJ—Wilhelm Sander-Stiftung (2017.130.1); ET—U.S. National Institutes of Health (GM127646).

**Acknowledgments:** The authors thank Aline Kraus and Yu-Shien Sung for assistance on the chemical characterization of EC1.

**Conflicts of Interest:** The authors declare no conflict of interest.