**4. Results**

#### **4.1. Frontal plane values**

#### *4.1.1. Mean distances between key anatomical landmarks in male and female subjects*

In the frontal plane, the mean distances between key anatomical landmarks can be seen in **Table 2**. The only statistically significant differences found between the two sides of the back in females were the distances between the acromion and the inferior scapular angle; with the right side distance (AR‐SR) being significantly smaller than the left side distance AL‐SL as seen in **Figure 2**. The other statistically significant difference found was between the inferior angle of the scapula and the apical thoracic vertebrae. For this parameter the right side dis‐ tance (TA‐SR) was significantly greater than the left sided value (TA‐SL). No other significant differences in the distances between key anatomical landmarks between the left and right sides of the back were found. Further no significant differences were found for all key ana‐ tomical landmark distances in male subjects between the two sides of the back. The mean female and male distances and standard deviations on the left and right sides of the back can be seen in **Tables 2** and **3**. The back landmarks positions and representation of key anatomical landmarks on a mannequin are presented in **Figures 3** and **4**. Normative values of analysed parameters can be seen in **Tables 4** and **5**.

**Table 2.** The distance between the acromion and the inferior scapula on the left side is statistically longer than the right side and the distance between the inf. angle of the scapula and the apical thoracic vertebrae on the right side is significantly longer on the right side than on the left side.

#### *4.1.2. Differences in female and male height values between the left and right sides of the back for key anatomical landmarks in the frontal plane*

The set of values below **Table 3** refers to the differences in height levels between specific key anatomical landmarks on the two sides of the back. The only significant differences found were at the level of the shoulders; The left shoulder acromion was significantly higher than the right (AL > AR). Otherwise no other staticatically significant differecnes were found at the levels of the inferior scapular angle, the iliac crests and the PSIS. In males there were no stat sign difference at any level, however at the PSIS level the left PSIS showed a trend toward being higher than the right PSIS


*4.3. Effect of the Concentration of Xanthate*

According to Equation (2), increasing the concentration of xanthate, i.e., the reactant, makes the reaction moves in the normal direction, resulting in more reaction product, i.e., CuX, produced on mineral surface. This concentration effect of xanthate is clearly shown in the AFM images obtained with the present work. For example, Figures 3, 4 and 6 clearly show that the amount of adsorbate increases greatly in surface coverage and the height of the adsorbate, when the concentration of KEX increases from 5 × 10−5 M to 5 × 10−4 M. The same conclusion can also be drawn for the case of PAX. By comparing Figures 8–10, one can see that when the concentration of PAX increases from 1 × 10−5 M to 1 × 10−4 M, the amount of adsorbate increases at a same contacting time.

## *4.4. Effect of Adsorption Time*

Figures 6, 7, 10 and 11 show that the height of the adsorbate and the surface coverage increased when the adsorption time increased from 10 min to 20 min in the xanthate solution at pH 6. In addition, as shown in Figure 12A–C and Figure 13A–C, the adsorption increased when the adsorption time increased from 5 min to 10 min and further to 20 min in the xanthate solution at pH 10. The trend was much more evident in the case of KEX. All these images clearly show that the adsorption of the xanthate on bornite increased with the adsorption time. The finding is in line with a common industrial practice of copper ore beneficiation, which involves adding collectors in a mill to increase the adsorption time, as well as the benefit from the adsorption of the collector on the freshly exposed mineral surface.

#### *4.5. Impact of Xanthate on Bornite Flotation*

In froth flotation, specific chemicals, i.e., collectors or promoters, are added to the pulp to increase the surface hydrophobicity of a target mineral. This results in the increase of both the attractive hydrophobic force and the adhesion force between the mineral particles and bubbles. The former can facilitate a particle/bubble attachment, and the latter can retard a particle/bubble detachment, which are both beneficial for froth flotation.

According to Cassie's equation [29]:

$$
\cos \theta \equiv f\_1 \cos \theta\_1 + f\_2 \cos \theta\_2 \tag{3}
$$

where *θ*1 is the contact angle for component 1 with a surface area fraction *f* 1, *θ*2 is the contact angle for component 2 with surface area fraction *f* 2 and *θ* is the contact angle of the composite material. In addition, *f* 1 + *f* 2 = 1 for the case of the adsorption of the collector on the bornite surface, assuming that component 1 is the bare bornite and component 2 is the adsorbate, i.e., CuX, *θ*2 should be larger than *θ*1. Therefore, increasing *f* 2 and/or *θ*2 will increase *θ*, i.e., the hydrophobicity of bornite with adsorbate, resulting in a better flotation.

In the present study, as shown in the AFM images, it is clear that xanthate can effectively adsorb on a bornite surface, as xanthate showed an almost full coverage at a concentration above 1 × 10−5 M PAX. This suggests that the adsorption of cuprous xanthate resulted in a large *f* 2, which is beneficial for a large *θ*. Increasing the dosage of xanthate and adsorption time will increase the cuprous xanthate's surface coverage, i.e., *f* 2, and it is also beneficial for to increase the surface hydrophobicity.

The force measurement results show that the detach forces measured in the xanthate solutions were larger than the force measured with water. In general, a large detach force suggests a large adhesion between a probe and substrate through the media. Following the Derjaguin approximation [30], it is predicted that a larger adhesion force will be achieved when the probe/liquid/bornite interfacial tension increases. In the present investigation, the Si3N4 AFM probe was inert in water, and it did not directly react with xanthate. Therefore, the surface energy of the Si3N4 probe did not change. In addition, in the present study, the surface tension of water media remained the same because the short-chain xanthate surfactant was used at a very low concentration. Therefore, the increase in the detachment force, as shown by the AFM force measurement, was mainly due to the increase of the interfacial tension between bornite and water, suggesting an increase in the hydrophobicity of bornite because of the adsorption of the cuprous xanthate on the mineral surface. We also suggest carrying out an AFM "colloid force" measurement by directly measuring the interaction force between a hydrophobic "colloid probe" and the adsorbate on bornite surface. The results will help to better understand the interaction between a bubble and a bornite particle in xanthate solutions in froth flotation. Such an investigation of force measurement, which is beyond the scope of the present study, is recommended for a future work.

#### **5. Conclusions**

AFM surface image measurements were applied to study the adsorption of xanthate on bornite in an aqueous solution in situ. The AFM images showed that the xanthate adsorbed on the mineral surface strongly when bornite contacted the KEX and PAX solution. The ATR-FTIR result confirms that that the adsorbate was essentially cuprous xanthate. Increasing the hydrocarbon chain length of xanthate increased the collectivity of the collector by increasing the surface coverage of the cuprous xanthate on the mineral surface at a lower concentration. Both increasing the chemical dosage and increasing the adsorption time will increase the surface coverage of CuX on mineral surface, which contributes to a better flotation by increasing the surface hydrophobicity of bornite.

**Funding:** This research received no external funding.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to ethical.

**Acknowledgments:** J. Zhang is grateful to Freeport-McMoRan Copper & Gold, Inc. for sponsoring the Freeport McMoRan Copper and Gold Chair in Mineral Processing in the Department of Mining and Geological Engineering in the University of Arizona. Reviewers' insightful comments and valuable suggestions are greatly appreciated.

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

#### **References**

1. Gaudin, A.M.; Schuhmann, R., Jr. The action of potassium n-amyl xanthate on chalcocite. *J. Phys. Chem.* **1936**, *40*, 257–275. [CrossRef]


**Abstract**

So far, many microwave dielectric materials have been investigated for a range of telecommunication applications. In dielectrics, the three main dielectric properties are quality factor (*Q*), dielectric constant and temperature coefficient of resonant frequency. Among these, the most essential dielectric property is *Q*. More specifically, *Q* is the inverse of the dielectric loss (tan*δ*); thus *Q* = 1/tan*δ*. There are two kinds of losses: those depending on crystal structure and losses due to external factors. The former is intrinsic losses such as ordering, symmetry, and phonon vibration. The latter is extrinsic losses due to factors such as grain size, defects, inclusions and distortion. In this chapter, the authors present the origin of dielectric losses based on the crystal structure. An ideal and well-proportional crystal structure constitutes a low loss material. Most dielectric materials are paraelectrics with inversion symmetry *i* and high symmetry. In general, it is believed that ordering gives rise to a high *Q*, on which many researchers are casting doubt. In the case of complex perovskites, the symmetry changes from cubic to trigonal. Ordering and symmetry should be compared with the structure. In this chapter, three essential conditions for the origin of high *Q* such as high symmetry, compositional ordering and compositional density are presented.

**Keywords:** microwave dielectrics, *Q*-factor, ordering, symmetry, indialite/cordierite, pseudo tungsten-bronze, complex perovskite


**4. Conclusion**

The representative PL decay curves for luminescence emission for the phosphors Ca0.5Y1-

are shown in **Figure 12a**, **b**. This can be fitted well into a single exponential function [15, 27]

t

where *I*0 is the luminescence intensity at times *t* = 0 and *τ* is its associated luminescence lifetime.

**Phosphor CCT (K) CRI Colour coordinates LER (lm W-1) Colour purity (%)** *τ* **(ms)**

Ca0.5Y(MoO4)2:Eu3+,Na+ 1149 33 0.635 0.365 162 90.0 0.462 Ca0.5Y(MoO4)2:Tb3+,Na+ N/A 26 0.296 0.564 491 87.5 0.455 Ca0.5Y(MoO4)2:Dy3+,Na+ 3658 18 0.414 0.478 525 81.3 0.172 Ca0.5La(MoO4)2:Eu3+,Na+ 1196 42 0.656 0.343 321 95.0 0.481 Ca0.5La(MoO4)2:Tb3+,Na+ 6850 12 0.251 0.570 530 91.9 0.485 Ca0.5La(MoO4)2:Dy3+,Na+ 4195 16 0.404 0.485 462 81.7 0.187

**Table 1.** Photometric parameters, color purity and luminescence decay time for the phosphors Ca0.5R1-*x*(MoO4)2:*x*Ln3+,

The thin phosphor films grown from nano-powder are being synthesized by the hydrothermal method, and the synthesis procedure is described previously by our group [17]. The luminescence emission intensity is being enhanced by co-doping of alkali metal ions. Furthermore, for the co-doping of alkali precursors, instead of using alkali chloride, alkali carbonates were taken and converted them into alkali nitrates. These alkali nitrates were co-doped with the existing precursors following the hydrothermal method nano-powders were synthesized and thin films were deposited from these powders [17]. The room temperature PL emission spectrum for Ca0.5R1-*x*(MoO4)2:*x*Eu3+,Na+ (R = Y, La) as the representative thin phosphor films are shown in **Figure 14**. The emission spectra monitored at 395 nm UV excitation for both the phosphors shows a number of intra-configurational ff transitions. The strong and most intense emission peak is found at 616 nm for Ca0.5Y1-

*x*(MoO4)2:*x*Eu3+,Na+ and 615 nm for Ca0.5La1-*x*(MoO4)2:*x*Eu3+,Na+ is attributed to the 5

electric-dipole transition possess hypersensitive red emission [15, 28, 29]. The Stark energy splitting is mildly shown for both the phosphors. It is noticed that the splitting of the electric dipole transition is uniform and homogeneous between the two thin phosphor films. The intensity of the spectral peaks for the nano-thin phosphor film is nearly close

**3.4. Photoluminescence emission studies from nano-architectures**

(Ln = Eu, Tb and Dy)

D0 → 7 F2

è ø (2)

(Ln = Eu, Tb and Dy) and Ca0.5La1-*x*(MoO4)2:*x*Ln3+,Na+

48 Applications of Laser Ablation - Thin Film Deposition, Nanomaterial Synthesis and Surface Modification

0exp *t I I*

The photometric quantities and luminescence decay time values are given in **Table 1**.

*x y*

æ ö - = ç ÷

*x*(MoO4)2:*x*Ln3+,Na+

as

Na+

(R = Y, La; Ln = Eu, Tb and Dy).

In conclusion, the nano-sized single crystalline Ca0.5La1-*x*(MoO4)2:*x*Eu3+,M+ ceramic thin phosphor films deposited on quartz substrates by pulsed laser deposition technique using Nd-YAG laser source in an ultra-high vacuum (UHV). The FESEM images exhibited the sphericalshaped phosphor particles. XRD patterns revealed the scheelite-type crystal structure without any impurity phases. By using AFM, the surface topographies and distributions of grains were investigated. Upon optical excitation, Eu-, Tb, and Dy-doped Ca0.5La1-*x*(MoO4)2:*x*Eu3+,M+ thin phosphor films showed characteristic emissions in the bright-red, green and yellow regions, respectively. The obtained results suggested that the deposited thin film phosphors could serve as efficient materials for electroluminescence and display applications.


**Author details**

Sílvia N.D. Maússe Sitoe and Joanna F. Dames\* Rhodes University, Grahamstown, South Africa

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Mitigating Climate Change: The Influence of Arbuscular Mycorrhizal Fungi on Maize… DOI: http://dx.doi.org/10.5772/intechopen.107128*


4.3.1. Formation Construction

In the formation construction experiments, eight mobile robots are deployed in the indoor scene. The robots are initialized at different positions with various environmental characteristics. We set the target formation of the robots as a rectangle; then, the eight robots will be trained together with the proposed Parallel Deep Deterministic Policy Gradient (PDDPG) algorithm. The hyperparameters of the PDDPG are listed in Table 2. In the training stage, we add the random bias to the target position values in each episode. It can enrich the formation data and reduce the overfitting problem of multi-robot training. Figure 5a,b illustrate one of the formation construction experiments in the testing stage. Figure 5a shows the initialization of the multi robot system; the robots are deployed in the indoor scene with various poses. Figure 5b shows the final results of the formation construction task. The curves with different colors represent the navigation trajectories of different mobile robots. As illustrated in the figures, all the mobile robots arrive at the target and construct the formation properly.

**Figure 5.** (**a**) the initial state of the group of mobile robots; (**b**) the terminal state of the group of mobile robots after accomplishing the formation construction navigation.

#### 4.3.2. Collaborative Navigation

After obtaining a good policy module in the formation construction task, we use it as the pretrained module in this section. The collaborative reward functions and the constraints are added in these experiments. As shown in Figure 6, the group of mobile robots can keep the rectangle formation during navigation. The trajectories of different mobile robots are illustrated by the colorful curves. In addition, the mobile robots at several timesteps are merged into one figure after getting the trajectory.

**Figure 6.** The trajectory of the multi-robot collaborative navigation.

To evaluate the versatility of the multi-robot navigation module, we also evaluate the module with the arrow formation. Figure 7 consists of four different figures, and they are recorded at the different timesteps. The intersection point of three colorful lines represents the origin of the environment. The group of robots navigated from the bottom left to the top right. This experiment illustrates that the mobile robots can keep the arrow formation during navigation.

**Figure 7.** The arrow formation of the multi-robot navigation system.

As illustrated in the collaborative navigation experiments, we mainly evaluate the formation keeping navigation tasks in the simulation environment without obstacles, since the distance of the obstacles can influence the performance of formation keeping. Generally, the multi-robot collaborative navigation task with formation keeping has two levels: one is the formation keeping navigation; the other is the multi-robot obstacle avoidance in a complex environment with constant formation. In this paper, we mainly focus on the former. In our work, the policy module of a collaborative navigation task utilizes the pre-trained policy module in the formation keeping task. There is lots of prior knowledge about obstacle avoidance that has been learned in the pre-trained module. Thus, the collaborative navigation module is sensitive about the obstacles. If there are obstacles in the navigation path, the formation of mobile robots can't keep the neat shape. The size of the obstacles can also influence the performance of formation keeping. In this paper, we mainly illustrate the formation keeping navigation tasks in the simulation environment without obstacles. In addition, we will address the multi-robot obstacle avoidance in a complex environment with constant formation keeping in future work.

To discuss the execution time of our method, we add the experiments to compute the execution time for the proposed Parallel Deep Deterministic Policy Gradient (PDDPG) algorithm. Since our end-to-end policy module can directly obtain the linear velocity and angular velocity through the raw sensor data, we can infer the execution time by computing the navigation velocity of the multi-robot system. There are two kinds of times in the Gazebo simulation platform: the simulation time and real time. The platform can accelerate the simulation or slow it down to fit specific tasks. To speed up the experimental efficiency, our work accelerates the simulation during training. Thus, we convert the simulation time to real time and compute the velocity of our multi-robot navigation system. As shown in Table 3, the group of robots navigates in four different kinds of trajectories with rectangular formation. The navigation distances, the time durations and the execution velocities are listed as follows. According to the results, we can infer that our method has an acceptable execution time.

**Table 3.** Experiments related to the navigation velocities of the multi-robot system.

#### **5. Conclusions**

This work mainly studied the collaborative formation and navigation of multi-robot system by using the deep reinforcement learning algorithm. By taking the raw 2D lidar sensor data and the relative target positions as inputs, the proposed Parallel Deep Deterministic Policy Gradient (PDDPG) algorithm could directly control the group of mobile robots to construct the formation and maintain it during navigation. The end-to-end policy module for mapless navigation was evaluated both in the

single-robot situation and the multi-robot situation. Our experimental results demonstrated that the proposed method could teach the multi-robot system to learn human intuition and accomplish the collaborative navigation tasks with a high arrival rate.

**Author Contributions:** Conceptualization, W.C. and S.Z.; Data curation, S.Z.; Formal analysis, S.Z.; Funding acquisition, Y.L.; Investigation, S.Z. and H.Z.; Methodology, W.C. and S.Z.; Project administration, W.C., S.Z. and Y.L.; Resources, Z.P. and H.Z.; Software, S.Z.; Supervision, Z.P. and Y.L.; Validation, W.C. and S.Z.; Visualization, W.C.; Writing—original draft, W.C.; Writing—review and editing, W.C.

**Funding:** This work is supported by the National Natural Science Foundation of China under Grant U1509210, 61836015.

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


**3.3 Verification of simulation method**

The purpose of this study is to quantitatively predict the residual stresses that are necessary before actual cylinder block for use. Therefore, it is important to set up an appropriate method of simulation, which is an economic evaluation method, before production. For this purpose, this study compares the surface pressure distribution of the simulation with the surface pressure prediction calculated as (**Figure 13** and Eq. 3) [6].

$$\mathbf{p} = \frac{\delta}{\frac{b}{E\_k} \left(\frac{b^2 + c^2}{c^2 - b^2} + \nu\_h\right) + \frac{b}{E\_\iota} \left(\frac{a^2 + b^2}{b^2 - a^2} - \nu\_\iota\right)}\text{ Face Pressure}\tag{3}$$

*Eh* ¼ modulus of elasticity for hole material,

*νh* ¼ Poisson0 s ratio for hole material,

*Es* ¼ modulus of elasticity for shrink material,

*νs* ¼ Poisson0 s ratio for shrink material

which is obtained by setting the following stress conditions (**Figure 14** and Eqs. (4) and (5)).

$$\frac{d\sigma\_r}{dr} + \frac{\sigma\_r - \sigma\_\theta}{r} = 0 \text{ Equilibrium Equation} \tag{4}$$

$$
\varepsilon\_r = \frac{du}{dr}, \varepsilon\_\theta = \frac{u}{r} \text{ Strain} \tag{5}
$$

*σr* ¼ radial stress,

*σθ* ¼ tangential stress,

*εr* ¼ radial strain,

*εθ* ¼ tangential strain,

*u* ¼ displacement of radial direction

Point 9: The left limit of the Haigh diagram is given by the ultimate stress limit

*Fatigue lines to various fracture theories. (a) Fracture theory, and (b) Yield theory.*

*New Challenges in Residual Stress Measurements and Evaluation*

However, the S-N curve and the S-S curve can be changed by the size effect, the relative stress gradient, and the temperature effect, which are exactly the values of

under compression of the material.

*Drawing method of Haigh diagram.*

**Figure 10.**

**Figure 11.**

**132**

**Figure 16.**

**Figure 17.**

**135**

*Relationship between rates of shrink fit and residual stress.*

*State of contact pressure and residual stress.*

*Durability Assessment Considering Residual Stress DOI: http://dx.doi.org/10.5772/intechopen.90298*

**Figure 13.** *State of shrink fit.*

**Figure 14.** *Stress state.*

**Figure 15.** *Check of surface pressure.*

*Durability Assessment Considering Residual Stress DOI: http://dx.doi.org/10.5772/intechopen.90298*

**Figure 16.**

*State of contact pressure and residual stress.*

**Figure 17.** *Relationship between rates of shrink fit and residual stress.*

**Figure 14.** *Stress state.*

**Figure 15.**

**134**

*Check of surface pressure.*

**Figure 13.** *State of shrink fit.*

*New Challenges in Residual Stress Measurements and Evaluation*

The resulting equation assumes axial symmetry and does not take into account shear stress inherently. In addition, the constant surface pressure in the longitudinal direction is calculated, so it is not suitable for the shape of the cylinder block which is the object of this study, but it is judged to be suitable as the relative standard of the surface pressure to be derived from the structure simulation.

a high residual stress value. Therefore, in this study, the durability assessment was performed considering the residual stress. In addition, in this section, realistic load and boundary conditions considering the use environment are examined to improve

The cylinder block is fixed from the shoe plate of the main pump and rotates about the rotation axis. At this time, the shoe plate has a certain angle, and the stroke of the piston is generated through this. As a result, during one revolution of the cylinder block, the working fluid flows into the piston through the top dead center and the bottom dead center, and the high-pressure operating fluid what the user wants is discharged [9]. At this time, the cylinder block can be divided into a low-pressure suction portion and a high-pressure discharge portion, which is a main working load condition acting on the cylinder block. In order to quantitatively confirm the magnitude of this high pressure, this study uses the swash plate angle of the main pump, the position of the piston end when the cylinder block is rotated and the AMESim program. As a result, realistic loads such as **Figures 19** and **20** were obtained.

In addition, the boundary condition as shown in **Figure 21** was set in consideration of the operating condition of the cylinder block. The boundary conditions were fixed in the axial direction where the stopper was located, and the angle of the spline portion was fixed to prevent rigid body motion of the cylinder block. In this case, the accuracy of the simulation is ensured by applying the centrifugal force to the influence of the rotational speed of the cylinder block while rotating the cylinder block.

the reliability of the structural simulation results.

**4.1 Simulation condition of cylinder block**

*Durability Assessment Considering Residual Stress DOI: http://dx.doi.org/10.5772/intechopen.90298*

**Figure 19.**

**137**

*Piston end position from bottom for simulation.*

#### **3.4 Estimate residual stress using simulation**

The residual stress of the derived cylinder block using the structural simulation is shown in **Figure 15**. In order to determine the suitability of the simulation results, the surface pressure of the bushing was confirmed by angle. The surface pressure distribution of the simulation results follows the general shape [7] of the Hertz contact theory and most of the length of the surface pressure is higher than the constant value derived from the numerical formula (Eq. 5). The reason is that the shape of the cylinder block is not perfectly symmetrical and shear stress is generated almost everywhere (**Figures 16** and **23**).

In addition, the shape and numerical value of the residual stresses were checked within the range of possible shrink fit to confirm the aspect of the change in the residual stress with respect to the rate of shrink fit [8]. As a result, as shown in **Figure 17**, the shape of the residual stress was less dependent on the rate of shrink fit, and the numerical value of the residual stress was almost linearly related to the rate of shrink fit. Therefore, it was possible to set the rate of shrink fit and the residual stress in a linear relationship within the range of the producible shrink fit. In this study, it was possible to select the rate of shrink fit optimized for the use environment by using this relationship. If the relationship between the rate of shrink fit and the residual stress was nonlinear, then the simulation was additionally performed to find out the sections having linear relationship to find the optimal design.


**5.3.42 Lecithin**

The term lecithin describes a group compounds consisting of phosphoric acid, choline, fatty acids, glycerol, glycolipids, triglycerides, and phospholipids. Typical members comprehend phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol.

In a clinical trial, lecithin did not show a benefit in ALS patients (Kelemen et al., 1982).


**1. Introduction**

Modern metallurgical installations such as steel plants and copper smelters require a range of plant sensors and process control systems to attain their highest efficiency. It is often stated that the levels of so-called "smart automation" today represents the fourth-generation industrial revolution (sometimes also called "Industry 4.0")—following the first, considered as the power generation and mechanical automation (early 1800s), the second as widespread industrialization (early 1900s), the third as electronic automation (starting 1950s), and, now, the fourth [1], benefitting from modern information and communication technology (ICT), as illustrated in Figure 1. Learning from history, it is well-known that if a plant does not remain up to date, taking advantage of modern but proven equipment and controls, then it lags its competitors. This paper discusses the application of techniques, sensors, and mathematical modeling that can support copper smelters in their endeavors to modernize their operations through data acquisition and, thus, remain competitive.

**Figure 1.** Representation of "Industry 4.0"—the 4th industrial revolution. Adapted from [1]. ICT: information and communication technology.

By way of background, copper smelters [2] are quite complex, with a number of individual high-performance operating units functioning together. Two of the key primary instrumentation and control needs in copper smelting today—and throughout history—are air (or oxygen) measurement and control and furnace temperature. In February 1956, a paper was presented at the AIME Annual Meeting in New York on the fine and well-recognized converter operation at the Noranda smelter (now Glencore Horne) located in Rouyn-Noranda, Quebec, Canada [3].

After describing how the production capacity at the fairly new plant increased over the previous two decades since the start-up (in 1927) by simply installing additional and/or larger units, the author discussed a major effort at further increasing production capacity that included the installation of reliable instruments for measuring both the instantaneous air flow and reporting the accumulated air blown over any given time interval. The significance and benefits of this new instrumentation (higher production rates) were subsequently described; of interest, the converters at this plant reached record or near-record air blowing rates in the industry at this time, reflecting the fact that it was one of the leading plants of the day. Of course, such equipment is mandatory today, coupled with the distributed control system (DCS) and advanced computer models to provide feedback for optimizing the operation.

Furnace temperature measurements at the time were made by an optical pyrometer mounted so that it pointed downward onto the molten bath. While generally reliable, it was often prone to dust and fume build-up affecting a clear sight line; unsmelted flux and other materials on the bath could also lead to low temperature readings. Some thirty years after the above noted paper [3], a new pyrometer instrument operated by sighting directly into the bath through one of the tuyères was described at the Copper 87 conference held in Viña del Mar, Chile [4]. During the discussion, the authors were asked about the cost of the new instrument. An indicative preproduction price range was given when the questioner loudly stated that he could buy "a Mercedes-Benz automobile" for the price mentioned, whereupon the authors responded that if the questioner installed one of these instruments at his plant, the profits he would make would allow him to purchase quite a few Mercedes! The point is that proper selection and use of reliable instruments for automated data acquisition and control are vital for a proper functioning plant to remain competitive, as the cost of sensors are comparatively modest.

In batch processes, such as steelmaking or copper converting, knowledge of the precise endpoint of the operation is extremely important for compositional control and optimization of the subsequent refining operations.

The Boliden's Rönnskär smelter, located at Skelleftehamn, Sweden, operates one of the best converter aisles in the world today, in the authors' opinion. They also pioneered the use of the ingenious Semtech Optical Process Control (OPC) sensor device for precise endpoint determination [5] to detect when the last of the iron was expelled into slag and, subsequently, when the last of the sulfur was expelled into the SO2 offgas. This instrument spectrometrically measures trace amounts of lead and copper sulfides and oxides in the offgas and signals when the level of sulfides starts to decrease, signaling the end of the batch [6]. The precise endpoint control saves significantly in processing time at Rönnskär in the subsequent anode furnace, thus lowering operating costs and allowing higher throughput.

In spite of the successful implementations of modern sensors at certain smelters such as Rönnskär, there is a general hesitance for copper smelters to adopt sensor arrays, as it is often unclear what the operational implications will be. Indeed, it is unclear what will be the series of changes that will be necessary so that the perceived benefits of the sensors will be manifest, and it is therefore difficult to quantify what will be the true impact of these new sensors. This paper presents a unified quantitative framework for copper and nickel-copper smelters, which integrate thermochemical modeling into discrete event simulations and is intended to assist in smelter reengineering projects that feature innovative sensors in consideration the Industry 4.0 paradigm.


*Review* **MicroRNAs as Predictive Biomarkers of Resistance to Targeted Therapies in Gastrointestinal Tumors**

**Valentina Angerilli 1,†, Francesca Galuppini 1,†, Gianluca Businello 1, Luca Dal Santo 1, Edoardo Savarino 2, Stefano Realdon 3, Vincenza Guzzardo 1, Lorenzo Nicolè 1, Vanni Lazzarin 1, Sara Lonardi 3, Fotios Loupakis 3 and Matteo Fassan 1,3,\***

**Abstract:** The advent of precision therapies against specific gene alterations characterizing different neoplasms is revolutionizing the oncology field, opening novel treatment scenarios. However, the onset of resistance mechanisms put in place by the tumor is increasingly emerging, making the use of these drugs ineffective over time. Therefore, the search for indicators that can monitor the development of resistance mechanisms and above all ways to overcome it, is increasingly important. In this scenario, microRNAs are ideal candidate biomarkers, being crucial post-transcriptional regulators of gene expression with a well-known role in mediating mechanisms of drug resistance. Moreover, as microRNAs are stable molecules, easily detectable in tissues and biofluids, they are the ideal candidate biomarker to identify patients with primary resistance to a specific targeted therapy and those who have developed acquired resistance. The aim of this review is to summarize the major studies that have investigated the role of microRNAs as mediators of resistance to targeted therapies currently in use in gastro-intestinal neoplasms, namely anti-EGFR, anti-HER2 and anti-VEGF antibodies, small-molecule tyrosine kinase inhibitors and immune checkpoint inhibitors. For every microRNA and microRNA signature analyzed, the putative mechanisms underlying drug resistance were outlined and the potential to be translated in clinical practice was evaluated.

**Keywords:** microRNAs; GI cancers; targeted therapy; drug resistance

### **1. Introduction**

The number of druggable tumor-specific molecular alterations has grown substantially in the past decade and a great survival benefit has been obtained from genomic-driven therapies across many cancer types. However, while molecularly targeted drugs offer a significantly higher response rate than traditional chemotherapy in diseases such as melanoma and non-small cell lung cancer, they have shown relatively modest clinical benefits in gastrointestinal (GI) malignancies [1]. The "one gene, one drug" approach of precision oncology clashes with the reality of the extremely complex molecular landscape of most solid tumors. A crucial aspect towards a successful development and application of targeted therapies is the understanding of resistance mechanisms that limit their effectiveness [1].

Primary or intrinsic resistance is defined as radiographic or clinical disease progression as the best response to an anticancer therapy and implies the pre-existence of resistancemediating factors within the tumor [2].

**Citation:** Angerilli, V.; Galuppini, F.; Businello, G.; Dal Santo, L.; Savarino, E.; Realdon, S.; Guzzardo, V.; Nicolè, L.; Lazzarin, V.; Lonardi, S.; et al. MicroRNAs as Predictive Biomarkers of Resistance to Targeted Therapies in Gastrointestinal Tumors. *Biomedicines* **2021**, *9*, 318. https://doi.org/ 10.3390/biomedicines9030318

Academic Editor: Francesca Lovat

Received: 7 February 2021 Accepted: 18 March 2021 Published: 21 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Secondary or acquired resistance is defined as therapeutic resistance after an initial period of disease stabilization or response, and be can caused by mutations arising during treatment, as well as through various adaptative mechanisms [3].

A wide range of biological determinants have been associated with drug resistance to targeted therapies; these include the presence of undruggable genomic drivers, the mutation of drug targets, the activation of survival signaling pathways, and the inactivation of downstream death signaling pathways. The tumor microenvironment may also mediate resistance by numerous mechanisms, including: promoting immune evasion of cancer cells, hampering drug absorption and stimulating paracrine cancer cell growth factors. The epithelial–mesenchymal transition (EMT) process and the presence of tumor stem cells have also been identified as causes of drug resistance. Furthermore, the coexistence of genetically and molecularly heterogeneous subclones within the bulk solid tumor may substantially fuel resistance under therapeutic selective pressure [2,4,5].

The purpose of this review is to elucidate the role of microRNAs (miRNAs) as mediators of resistance to targeted therapies in GI tumors.

#### **2. MicroRNAs and Drug Resistance**

MiRNAs are a group of short non-coding RNA transcripts (18–22 nucleotides) that control gene expression at the post-transcriptional level, thus regulating several pathways involved in the maintenance of cell homeostasis. In most cases, they act by binding to the 3 -untranslated region (3 -UTR) of their target messenger RNAs (mRNAs), which results in mRNA silencing or degradation and subsequent post-translational degradation or down-modulation of proteins [6,7].

Numerous studies have demonstrated that miRNAs are heavily dysregulated in cancer and in certain conditions they may function as oncogenes or tumor suppressor genes. Hence, microRNAs have been identified as potential diagnostic, prognostic and predictive biomarkers for patient stratification in oncology [8,9]. Particularly, miRNAs have been found to act as mediators of drug resistance through various cellular and molecular mechanisms, which are related to: apoptosis, cell cycle modification, alteration in drug targets, regulation of drug efflux transporters, epithelial-mesenchymal transition (EMT) and cancer stem cells [10–12] (Figure 1).

**Figure 1.** MiRNAs and mechanisms of resistance to targeted therapies in GI tumors. Credits to BioRender.com, accessed date 18 March 2021.

In light of their high stability in tissues and bodily fluids (i.e., plasma and urine), miRNAs are ideal candidates to monitor cancer treatment resistance. In fact, miRNAs are not only present within cells, but are also actively secreted from cells, and included in RNA-binding multiprotein complexes and/or exosomes [13]. In the context of liquid biopsies, analysis of miRNA expression patterns represents a promising tool to perform non-invasive longitudinal tracking of drug sensitivity over time [14,15].

When referring to GI malignancies, the role of miRNAs as predictors of resistance to chemotherapy has been extensively studied [16–18], while further research is needed to gain a better understanding of the complex interplay between miRNA expression and resistance to targeted therapy. Furthermore, many studies have documented the change in miRNA expression profiles following radiotherapy, so that miRNAs could prove useful in modulating radioresponsitivity at the clinical level [19].

With regard to future perspectives, playing such an important role in carcinogenesis, miRNAs have also been proposed and tested as a therapeutic option. In cancer, in order to increase the level of a tumor suppressor miRNA, whose level is downregulated, miRNA mimics can be used to re-establish normal expression and function of a certain miRNA of interest. On the other hand, antagomiRs can be used to suppress the function of a specific oncomiRNA that play a role in the development and progression of the neoplasm. However, while many studies have attempted to evaluate the consequences of the manipulation of these molecules with the purpose to support findings from in vitro or in vivo models, only a few miRNA targeted drugs have entered clinical trials because of a few important limitations, such as dosage and cross-reactions with other miRNAs [20].

#### **3. Anti-EGFR**

Epidermal growth factor receptors (EGFRs) are a large family of receptor tyrosine kinases expressed in several types of cancer and are responsible for the activation of a downstream signaling cascade that modulates growth, signaling, differentiation, adhesion, migration and survival of cancer cells [21].

Targeting EGFR has been revealed to be a successful strategy in metastatic colorectal cancer (mCRC). Cetuximab and panitumumab are the two anti-EGFR antibodies currently in use for mCRC [22]. Anti-EGFR antibodies act by binding the extra-cellular domain of EGFR, thereby preventing ligand binding and blocking the downstream signaling pathway. Furthermore, they induce receptor internalization and degradation [23], and are responsible for antibody-dependent cellular cytotoxicity [24].

In patients with metastatic disease, anti-EFGR monoclonal antibodies have shown a clinically significant response when given in monotherapy or in combination with chemotherapy [25,26]. However, drug resistance frequently arises. Many genomic and nongenomic mechanisms underlie resistance to anti-EGFR therapy. Particularly, a plethora of overlapping de novo and acquired genetic alterations have been identified, such as *KRAS*, *NRAS*, *BRAF*, *PIK3C,* and *EFGR* mutations and *MET* and *ERBB2* amplifications [27,28].

*Let-7. KRAS* mutations are the major predictors of resistance to EGFR inhibitors and are routinely screened in mCRCs [29]. In this regard, members of the Lethal-7 (Let-7) family of miRNAs have been found to display KRAS downregulating activity by binding to specific sites in the 3 -UTR of KRAS mRNA [30]. Ruzzo et al. measured the expression of let-7 in formalin-fixed paraffin-embedded (FFPE) tumor samples of mCRC patients who underwent third-line therapy with cetuximab plus irinotecan. The findings of this study revealed that higher levels of let-7 were significantly associated with better survival outcome in patients, regardless of *KRAS* mutational status. In patients with *KRAS* mutations, let-7 may serve to identify a subpopulation of responders to anti-EGFR therapy [31]. Furthermore, Cappuzzo et al. found that the high-intensity signature of the cluster Let-7c/miR-99a/miR-125b is associated with a favorable response to cetuximab and panitumumab in *KRAS* wild-type mCRC patients [32].

*MiR-31-5p/miR-31-3p.* The predictive role of miR-31-5p and miR-31-3p has been investigated in several studies. In a study which included an exploratory cohort and a validation

cohort, of the 9 miRNAs analyzed in FFPE tumor samples of both cetuximab responders and non-responders, miRNA-31-3p and miRNA-31-5p were found to be strongly associated with time to progress (TTP) in wild-type *RAS* patients treated with cetuximab [33]. In another study it was further discovered that high miR-31-5p expression was associated with shorter progression-free survival (PFS) in mCRC patients treated with anti-EGFR therapy [34]. Furthermore, Manceau et al., after screening 1145 miRNAs on a large cohort of wild-type *RAS* mCRC patients, identified miR-31-3p as a promising predictive biomarker of response to anti-EGFR antibodies [35].

*MiR-10/miR-125b.* MiR-10 and miR-125b are generated by the long non-coding RNA (lncRNA) MIR100HG, and decrease the expression of five negative regulators of the Wnt/βcatenin pathways, resulting in increased Wnt signaling, which is a hallmark of colorectal carcinogenesis. It was demonstrated that the overexpression of lncRNA MIR100HG and the two embedded miRNAs was associated with cetuximab resistance in tumors derived from CRC patients and in vitro 3-D cell culture models [36].

*MiR-181a.* MiR-181a has a strong tumor-promoting effect via inhibition of the tumor suppressor factor Wnt Inhibitory factor 1 (WIF-1) and stimulates tumor growth, cell motility and invasion. Pichler et al. analyzed the expression of miR-181a in FFPE tumor samples of a cohort of patients with *KRAS* wild-type mCRC, undergoing cetuximab and panitumumab treatment. The findings of the study highlighted the potential predictive role of miR-181a, as high levels of this miRNA were associated with a longer PFS [37].

*MiR-345.* According to the results of a study by Schou and colleagues, high levels of miR-345 in whole blood correlated with a lack of response to therapy in patients treated with third-line cetuximab and irinotecan [38]. In the context of liquid biopsy, miR-345 appears to be a valid candidate biomarker of sensitivity to anti-EGFR therapy.

*MiR-199a/miR-375.* Mussnich et al. performed miRNA expression profiling of human CRC cell lines sensitive to cetuximab and of their resistant counterparts. Among the investigated miRNAs, miR-199a and miR-375 were found to be overexpressed in resistant cells and their upregulation promoted cetuximab resistance. MiR-199a and miR-375 both target PH Domain And Leucine Rich Repeat Protein Phosphatase 1 (PHLPP1), which acts as a tumor suppressor by downregulating the Akt oncogenic pathway [39].

*MiR-302.* In a recent study, downregulation of miR-302a was observed in cetuximabresistant CRC cells as well as in patient-derived xenografts. Furthermore, miR-302a overexpression restored cetuximab responsiveness in CRC cell lines both in vitro and in vivo. MiR-302a has been found to act as a tumor suppressor by targeting NFIB (Nuclear factor 1 B-type) and downregulating the NFIB/ITGA6 axis, which is responsible for cell migration and invasion in the metastatic process [40].

*MiR-141-3p.* The findings of a study by Xing and colleagues indicated that miR-141-3p might be a predictor of response to cetuximab. In CRC cell cultures, miR-141-3p regulated cetuximab sensitivity by directly targeting EGFR and its downstream cascade. Moreover, miR-141-3p improved cetuximab-induced apoptosis in CRC cells, proving to have a tumor-suppressing function [41].

#### **4. Anti-VEGF**

Vascular endothelial growth factor (VEGF) promotes tumor angiogenesis through several mechanisms, including enhanced endothelial cell proliferation, survival and migration, chemotaxis of bone-marrow-derived progenitor cells, vascular permeability, and vasodilation. VEGF ligand acts by binding the VEGF receptor (VEGFR) with tyrosine kinase activity, which activates a network of downstream signaling pathways, responsible for mediating numerous changes within the tumor vasculature. Several strategies have been put in place to inhibit the VEGF pathway, including monoclonal antibodies blocking VEGF or VEGFR, soluble VEGF receptors, and tyrosine kinase inhibitors selective for VEGFRs [42].

Bevacizumab is a monoclonal antibody directed against VEGF and has been approved in combination with cytotoxic chemotherapy as first or second-line therapy in mCRC, after several randomized clinical trials had shown improvements in overall survival [43–45]. The VEGF decoy receptor aflibercept has also recently been introduced in clinical practice as second-line therapy in mCRC [46].

In the context of gastro-esophageal malignancies, the anti-VEGFR2 antibody ramucirumab is currently licensed for clinical use in combination with cytotoxic chemotherapy for chemo-refractory metastatic disease [47,48]. Sunitinib and sorafenib are multi-tyrosine kinase inhibitors (TKIs), which target a wide spectrum of pathways involved in tumor growth, including VEGF/VEGFR, and are in use in GI precision oncology.

However, the VEGF pathway inhibitors are failing to provide a durable response in most patients. The activation of alternative angiogenic "escape" pathways is a major contributor to drug resistance and may induce tumor growth, enhancement of invasion and metastasis [49]. A few preliminary studies have been carried out to elucidate the role of miRNAs as mediators of resistance to bevacizumab, however there is still no evidence regarding ramucirumab or aflibercept.

*MiR-126.* MiR-126 acts by enhancing the proangiogenic actions of VEGF and promotes blood vessel formation and survival [50]. MiR-126-containing micro-vesicles originate from endothelial cells and fuse with the membrane of neighboring cells, thus promoting angiogenesis in a paracrine manner. A negative relationship exists between levels of circulating miR-126 and their response to bevacizumab. Increasing plasma levels of miR-126 are associated with resistance to bevacizumab, while lower levels indicate treatment response. As miR-126 is representative of endothelial cell turn-over, its circulating levels could help perform non-invasive monitoring of bevacizumab resistance.

*MiR-664-3p/miR-455-5p*. The predictive role of miR-664-3p and miR-455-5p has been evaluated in a study by Boisen et al. Higher miR-664-3p and lower miR-455-5p expression evaluated in FFPE tumor samples was found to be associated with an improved outcome in the cohorts of patients treated with bevacizumab and chemotherapy, in comparison with the cohort treated with chemotherapy only. Therefore, miR-664-3p, with its putative tumor suppressor role, and miR-455-5p, with its putative oncogenic role, could represent potential predictive tissue biomarkers of bevacizumab effectiveness [51].

*MiR-20b-5p/miR-29b-3p/miR-155-5p.* According to the results of a study by Ulivi and colleagues, higher circulating levels of miR-20b-5p, miR-29b-3p and miR-155-5p are associated with a better outcome in mCRC patients treated with a combination of bevacizumab and chemotherapy. Furthermore, the variation in plasma levels of miR-155-5p could also be indicative of patient survival. In line with these findings, previous studies have found that miR-20b, miR-29b and miR-155-5p play a role in regulating tumor angiogenesis [52]. MiR-29b acts as a tumor suppressor through simultaneously inhibiting angiogenesis and tumorigenesis by targeting Akt3 [53]. Although little information is available on its involvement in the tumor angiogenic process, miR-20 has been found to regulate proliferation and senescence in endothelial cells [54]. MiR-155 contributes to controlling hypoxia-inducible factor (HIF-1α) and promotes angiogenesis under hypoxia condition [55].

#### **5. Anti-HER2**

HER2 belongs to the EGFR tyrosine kinase family. It is localized in the cell membrane and when activated initiates intracellular downstream signaling involved in diverse biological processes related to cancer, such as proliferation, migration and apoptosis [56].

Overexpression of HER2 has been detected in 11–20% of gastric and gastroesophageal junction (GEJ) cancers [57–60]. Trastuzumab is an anti-HER2 antibody that acts by blocking the activity of the HER2 receptor and weakening the downstream signaling [61].

In 2010, the results of the Trastuzumab for Gastric cancer (ToGA) trial displayed how a combination of trastuzumab and chemotherapy was able to prolong the overall survival compared to chemotherapy alone in gastric cancer patients with HER2 overexpression. Thus, trastuzumab in combination with chemotherapy has been approved as the standard first-line treatment of advanced gastric or GEJ cancer with HER2 overexpression/*ERBB2* amplification [62]. However, only a fraction of the patients has been found to respond

to trastuzumab, and even those who achieved an initial therapeutic response developed resistance within 7 months [63].

*MiR-21.* It has been found that trastuzumab activates phosphatase and tensin homolog (PTEN) phosphatase, thus decreasing PTEN tyrosine phosphorylation, via inhibition of HER2 receptor bound Src. Reduced PTEN expression could predict trastuzumab resistance [64]. According to the results of a study by Eto et al., overexpression of miR-21 downregulates PTEN and increases the phosphorylation of PTEN downstream target Akt in HER2-positive gastric cancer (GC) cell lines, thus resulting in decreased sensitivity to trastuzumab-induced apoptosis. The opposite effect in PTEN and p-Akt was observed after miR-21 suppression, which instead restored resistance to trastuzumab. These findings suggest that the miR-21/PTEN pathway might play a critical role in regulating trastuzumab resistance of GC cells via modulating apoptosis [65].

*MiR-223.* F-box and WD repeat domain-containing 7 (FBXW7) is the substrate recognition component of an evolutionarily conserved ubiquitin ligase complex, which appears to have an important role in controlling the stability of several oncoprotein substrates, including cyclin E, c-Myc, Notch, c-Jun, mammalian target of rapamycin (mTOR) and Myeloid Cell Leukemia 1 (MCL1) [66]. Upregulation of miR-223 decreased FBXW7 expression and subsequently reduced the sensitivity of HER2-positive GC cell lines to trastuzumab, thereby acting as an oncomiR and suppressing trastuzumab-induced apoptosis. On the contrary, downregulation of miR-223 restored FBXW7 expression and the sensitivity to trastuzumab [67].

*MiR-16.* Trastuzumab has the ability to block PI3K/AKT downstream signaling, which results in the inhibition of c-Myc activation and subsequent upregulation of miR-16. A study by Venturutti et al. identified cyclin J and Far Upstream Element Binding Protein 1 (FUBP1) as miR-16 targets. miR-16 acts as a tumor suppressor by exerting an antiproliferative effect via silencing its miRNA targets [68]. Furthermore, it was found that in vitro overexpression of miR-16 and low or null levels of FUBP1 were predictors of trastuzumab sensitivity. These findings suggest that both miR-16 and FUBP1 could represent promising predictive biomarkers of response to trastuzumab [69].

*MiR-125b.* MiR-125b has been found to be dysregulated in several cancer types. For example, while being downregulated in osteosarcoma, breast cancer, ovarian cancer and hepatocellular carcinoma (HCC), it appears to be upregulated in CRC, prostate cancer, non-small cell lung cancer and GC [70]. In the latter, miR-125b functions as an oncogene by promoting cellular proliferation, migration and invasion by downregulating the expression of Protein Phosphatase 1 Catalytic Subunit Alpha (PPP1CA) and upregulating Rb phosphorylation. Furthermore, it has been recently demonstrated that high miR-125b levels are associated with poor prognosis in patients with HER2-positive GC treated with trastuzumab, thus indicating a speculative role of miR-125b in trastuzumab resistance [71].

*MiR-200c.* Compelling evidences identify in the EMT one of the mechanisms implicated in drug resistance [72]. In a recent study by Zhou et al. it was observed that trastuzumab-resistant GC cell cultures expressed high levels of EMT markers, as well as TGF-β, which is a master regulator of the EMT. Furthermore, while miR-200c was found to be downregulated in trastuzumab-resistant GC cells, thereby proving to act as a tumor suppressor miRNA, its overexpression restored trastuzumab sensitivity and blocked the EMT by targeting Zinc finger E-box-binding homeobox 1 (ZEB1) and 2 (ZEB2), which are downstream molecules of TGF-β. This study suggested that the TGF-β/ZEB/miR-200c axis is involved in the resistance of trastuzumab in GC by regulating the EMT [73].

*MiR-494.* Lapatinib is a small molecule which inhibits the tyrosine kinases of HER2 and EGFR1 [74]. MiR-494 has been found to act as a tumor suppressor and to restore lapatinib sensitivity and inhibit formation of cancer-initiating cells (CICs) via reducing expression of FGFR2 in HER2-positive, FGFR2 overexpressing and lapatinib resistant GC cell cultures [75].

#### **6. Small-Molecule Tyrosine Kinase Inhibitors**

Receptor tyrosine kinases (RTKs) are widely expressed transmembrane proteins that regulate many fundamental cellular processes, and play a key role in the physiopathology of several diseases. Upon ligand binding, RTKs activate intracellular signaling pathways involved in many functions, such as: differentiation, proliferation, migration, invasion and angiogenesis. Aberrant RTK expression being a well-recognized mechanism of tumorigenesis, a broad variety of inhibitors are currently in clinical use across many cancer types [76]. Particularly, small-molecule tyrosine kinase inhibitors act by blocking the intracellular domain of the receptor or by inhibiting the tyrosine kinase activity of downstream signaling mediators [77].

#### *6.1. Imatinib*

Imatinib is a selective inhibitor of certain tyrosine kinases and is highly active in patients with gastrointestinal stromal tumors (GISTs) by blocking the constitutive activity of KIT and platelet-derived growth factor receptor α (PDGFR-α). Imatinib is the treatment of choice for advanced GISTs harboring *KIT* and *PDGFR-α* mutations, and it is also used in adjuvant and neoadjuvant settings. Although imatinib has high response rates, drug resistance remains the main challenge for extending patient survival [78]. The mechanisms underlying imatinib resistance are not completely clear. However, in about half of patients, resistance is caused by secondary mutations in *KIT* (exons 13, 14, 17 or 18) [79].

*MiR-125a-5p.* In a study on FFPE GIST samples from patients resistant to imatinib in a neoadjuvant setting, it was found that miR-125a-5p can modulate imatinib response in *KIT*-mutated GIST cells by regulating the expression of Protein Tyrosine Phosphatase Non-Receptor Type 18 (PTPN18). Because the targets of miR-125a-5p have been functionally associated with anti-apoptosis, cell cycle progression, signal transduction and protein phosphorylation, miR-125a-5p acts as oncomiR and its overexpression was linked with imatinib resistance [80].

*MiR-320a.* miR-320 acts as a tumor suppressor by targeting genes involved in Wnt and IGF pathways. In imatinib resistant GIST patients, down-regulation of the tumor suppressor miR-320a is associated with direct up-regulation of β-catenin and subsequent enhanced expression of anti-apoptotic MCL1 [81].

*MiR-218.* According the findings of a study by Fan and colleagues, the expression of miR-218 was down-regulated in an imatinib-resistant GIST cell line, whereas miR-218 overexpression was able to restore the sensitivity of GIST cells to imatinib, with the PI3K/AKT signaling pathway possibly involved in the mechanism. The PI3K/AKT pathway is downstream of KIT and it is reactivated when GISTs become resistant to imatinib [82].

*MiR-518a-5p.* By performing a microarray analysis on GIST samples from patients resistant to imatinib, miR-518a-5p was found to be a potential predictor of drug sensitivity. Downregulation of miR-518a-5p is likely to upregulate PIK3C2A, causing resistance to imatinib in GISTs. PIK3C2A belongs to the phosphoinositide 3-kinase (PI3K) family, whose member proteins have roles in signaling pathways involved in cell proliferation, oncogenic transformation, cell survival, cell migration and intracellular protein trafficking [83].

*MiR-92a-3p, miR-99a-5p, and miR-101-3p.* MiR-92a-3p, miR-99a-5p and miR-101-3p are three miRNAs implicated in cell cycle regulation. MiR-92a-3p targets Cyclin-dependent kinase inhibitor 1C (CDKN1C), miR-92a-3p regulates mTOR pathway [84], and miR-101-3p also acts as a regulator of the mTOR pathway by mediating AKT activation [85]. The findings of a study, which analyzed miRNA expression profiles across a series of imatinib resistant and sensitive FFPE GIST samples, identified the previously mentioned miRNAs as differentially expressed and therefore possibly implicated in imatinib resistance [86].

*MiR-28-5p.* A recent study identified miR-28-5p as a potential mediator of imatinib resistance in GISTs. Being overexpressed in imatinib-resistant GIST samples and displaying a significant correlation to imatinib response, miR-28-5p has been proven to function as an oncomiR. However, very little is known about this miRNA and therefore further research is required to confirm these findings [87].

#### *6.2. Sorafenib*

Sorafenib is a small molecule which inhibits multiple kinases involved in tumor cell signaling, proliferation, angiogenesis and apoptosis. Sorafenib is currently in clinical use for unresectable HCC. Its approval was based on the successful outcome of the pivotal SHARP and Sorafenib Asia-Pacific trials in Child-Pugh class A patients with advanced HCC. However, only approximately 30% of patients clinically benefit from sorafenib, and this subgroup usually acquires drug resistance within 6 months. Recent studies have highlighted the role of epigenetics, transport processes, regulated cell death, and the tumor microenvironment in the initiation and development of sorafenib resistance in HCC [88].

*MiR-122.* MiR-122 is the most abundant liver-specific miRNA and it is significantly down-regulated in HCC. miR-122 acts as a tumor suppressor in the liver by inhibiting survival of cancer cells, anchorage-independent growth, migration, invasion, and EMT. The in vitro restoration of expression of miR-122 has been found to sensitize HCC cells to sorafenib. MiR-122 is a negative regulator of A Disintegrin and metalloproteinase domaincontaining protein 10 (ADAM10) and serum response factor (SRF), which are both involved in EMT (SFR is also implicated in tumor angiogenesis) and insulin growth factor 1 receptor (IGF1R), which activates the downstream RAS/RAF/ERK pathway, induces proliferation and promotes metastasis [89].

*MiR-34.* In a study by Yang and colleagues, miR-34 was reported to be downregulated in FFPE HCC samples from patients and HCC cell lines and was associated with poorer survival. MiR-34, which is a direct target of p53, binds the 3 -UTR region of the antiapoptotic protein B-cell lymphoma 2 (Bcl-2), which was found to be overexpressed in HCC samples and the cells analyzed. The restoration of miR-34a potentiated sorafenib-induced apoptosis, suggesting that miR-34a enhanced the anti-tumor effect of sorafenib in HCC cells [90].

*Let-7.* To assess the role of miRNAs in HCC, Ohta et al. performed microarray analysis and discovered that let-7 as was downregulated in human HCC cells. Furthermore, it was also found that the upregulation of let-7 was linked to a decreased expression of its putative target, the anti-apoptotic protein B-cell lymphoma xL (Bcl-xL). Ultimately, the expression of let-7c enhanced apoptosis of HCC cells upon exposure to sorafenib, which is responsible for the downregulation of another anti-apoptotic Bcl-2 protein, MCL1. To summarize, let-7 exerts a tumor suppressing function by inducing apoptosis of HCC cells [91].

*MiR-338-3p.* Hypoxia being one of the main contributors to anti-tumor drug resistance in solid malignancies, hypoxia-inducible factor 1 (HIF-1) has been recognized as one of the key mediators of resistance to sorafenib in HCC. In a study on both patient samples and HCC cell lines, it was discovered that miR-338-3p directly targeted HIF-1α and downregulated the expression of HIF-1α target genes involved in the hypoxia-induced signaling pathway. Via this mechanism, miR-338-3p inhibits HCC tumor growth and sensitizes HCC cells to sorafenib [92].

*MiR-216a/217.* According to the findings of a study by Xia and colleagues on a series of HCC FFPE samples, chemoresistance against sorafenib is caused by overexpression of the miR-216a/217 cluster. Upregulation of miR-216a/217 is able to induce EMT of cancer cells and decrease expression of SMAD7 and PTEN, consequently activating the transforming growth factor β (TGF-β) and PI3K/AKT pathways [93].

*MiR-93.* Following a comprehensive miRNA expression profiling using HCC cell lines, miR-93 was identified as a novel target associated with HCC. MiR-93 acts by binding with the 3 -UTR of PTEN and cyclin-dependent kinase inhibitor (CDK1NA), inhibiting their expression and, as a result, activating the oncogenic PI3K/AKT pathway. In light of these findings, miR-93 expression was also found to render HCC cells more sensitive to sorafenib [94].

*MiR-21.* As previously mentioned, miR-21 dysregulates PTEN, by inhibiting Akt activation. The Akt signaling pathway appears to be highly activated in sorafenib-resistant HCC cells. Moreover, autophagy seems to promote sorafenib sensitivity in sorafenibresistant HCC cells. Compelling evidence suggests that miR-21 plays a key role in mediating resistance to sorafenib. For example, exposure of HCC cell lines to sorafenib led to an upregulation of miR-21 and a downregulation of PTEN. Furthermore, transfection of miR-21 in HCC cells was able to restore sorafenib resistance by inhibiting autophagy. To summarize, miR-21 contributes to acquired resistance to sorafenib by suppressing autophagy by modulating the Akt/PTEN pathway [95].

*MiR-193a.* MiR-193a acts a tumor suppressor by negatively regulating the prometastatic factor urokinase-type plasminogen activator (uPA) and is downregulated in HCC. Transfection of HCC cell lines with miR-193a was found to decrease proliferation, promote apoptosis and enhance sorafenib anti-tumor activity [96].

*MiR-193b.* Substantial downregulation of miR-193b and overexpression of its target MCL1 were observed in HBV-positive HCC cells. MCL1 is an anti-apoptotic protein, which is overexpressed in many human malignancies, including HCC, and has been known as an important mediator of chemosensitivity in HCC. Restoration of the expression of miR-193b sensitized HBV-positive HCC cell lines to sorafenib by facilitating sorafenib-induced apoptosis [97]. Similarly, in another study, HCV-positive HCC cells showed decreased expression of miR-193b and upregulation of its target MCL1 [98].

*MiR-221.* The results of a study by Fornari and colleagues indicated that in both HCC cell lines and xenografts, miR-221 overexpression was linked with sorafenib resistance, by targeting caspase-3 and thereby exerting an anti-apoptotic and oncogenic function. Moreover, when investigating the putative role of miR-221 as a circulating biomarker in HCC patients, lower pre-treatment circulating miR-221 levels were detected in sorafenib responders [99].

*MiR-486.* In HCC tissues and cell lines, miR-486 appears to function as a tumor suppressor by targeting *CITRON* and *CLDN1*, two genes which are responsible for the regulation of cell proliferation and invasion. In addition to being downregulated in HCC, miR-486 was found to enhance chemosensitivity of HCC cells to sorafenib [100].

*MiR-494.* In a recent study by Pollutri et al., miR-494 overexpression was reported to enhance sorafenib resistance via mTOR pathway activation both in vitro and in vivo. In fact, p27, PTEN and p53-upregulated-modulator-of-apoptosis (PUMA) were identified as targets of miR-494, contributing to speed up cell cycle progression, survival and invasiveness. Moreover, high miR-494 expression seemed to be associated with stem-like features [101].

*MiR-101.* As part of the tumor microenvironment, HCC-associated macrophages accelerate tumor progression by releasing growth factors. High TGF-β expression in M2 polarized macrophages is thought to increase tumor growth, metastases and EMT. In HCC cell lines, it was observed that miR-101 targeted dual specificity phosphatase 1 (DUSP1), inhibited TGF-β activation and enhanced the effect of sorafenib in HCC cells by potentiating macrophage modulation of the innate immune responses [102].

#### *6.3. Regorafenib*

Regorafenib is small-molecule multiple kinase inhibitor. In mCRC, it is indicated for patients who have been previously treated with, or are not considered candidates for, available therapies, including chemotherapy, an anti-VEGF therapy and, if RAS wild-type, an anti-EGFR therapy [103]. It is also licensed as second-line therapy for advanced HCC patients [104] and as third-line therapy for metastatic or unresectable GIST patients [105].

Despite the observed survival benefits, resistance to regorafenib is fairly common. Moreover, toxicity is not insignificant and the absolute clinical benefit is rather small. Therefore, it is crucial to identify efficient predictive biomarkers in order to optimize the use of regorafenib [106].

*MiR-34a.* In a recent study on CRC cell lines, Cai et al. showed that regorafenib is able to reduce stemness and tumorigenesis in vitro, by upregulating the tumor suppressor miR-34, which targets the WNT/β-catenin pathway. Thereby it can be inferred that regorafenib is able to suppress the generation of drug-resistant cancer stem-like cells via modulation of miR-34a-associated signaling [107].

*MiR-30a-5p.* Signal transducer and activator of transcription 3 (STAT3) is a transcriptional factor which contributes to drug resistance in cancer therapies by promoting tumor growth and cancer stemness. miR-30a-5p is a STAT3 downstream miRNA and targets Heat Shock Protein Family A Member 5 (HSPA5), which is a master regulator of unfolded protein response (UPR). A dysregulation of the STAT3-miR-30a-5p-HSPA5 axis, with a downregulation of miR-30a-5p, was observed in regorafenib-resistant in CRC tumorspheres [108].

In the context of liquid biopsies, a few preliminary studies have been carried out to investigate putative circulating predictive biomarkers. In a recent study by Schirripa and colleagues, an miRNA signature involving c-miR-21, c-miR-221 and c-miR-760 was found to be prognostic and predictive of a response to regorafenib in an exploratory cohort of CRC patients. However, the results were not confirmed by the validation cohort [103]. In another study on a large cohort of HCC patients, of the 750 miRNAs analyzed, increased plasma levels of miR-30a, miR-122, miR-125b, miR-200a, and miR-374b decreased levels of miR-15b, miR-107 and miR-320b, and absence of miR-645 were all predictive of survival benefit with regorafenib [109].

#### **7. Immune Checkpoint Inhibitors**

The introduction of immune-checkpoint blockades in precision oncology led to a paradigm change in the management of advanced cancers. The rationale behind these novel therapies is that cancer cells are able to evade immunosurveillance through different mechanisms, including activation of immune checkpoint pathways that suppress antitumor immune responses [110].

Immunotherapy has recently been incorporated in treatment regimens of GI malignancies. The immune-checkpoint inhibitors currently in clinical use in GI oncology are nivolumab and pembrolizumab, which target programmed death-1 (PD-1) and ipilimumab, which targets cytotoxic T-lymphocyte antigen-4 (CTLA-4) [111].

Immunotherapy approaches have been extensively studied in CRC. In the metastatic setting, ICI therapies provide clinical benefit to defective mismatch repair and microsatellite instable (dMMR/MSI) tumors. Pembrolizumab has been approved as first-line treatment in dMMR/MSI mCRCs based on the successful outcomes of the KEYNOTE-177 trial [112].

As with gastroesophageal cancers, pembrolizumab is currently licensed for recurrent locally advanced or metastatic cancers with PD-L1 expression [113]. Much like in CRC, MSI gastric cancers greatly benefit from ICIs [114]. Moreover, EBV-positive GCs have shown an even more promising response to pembrolizumab [115]. ICIs have also been recently approved as second-line therapy in HCC, after a single agent checkpoint blockade trials obtained partial success [116].

However, a subset of patients who initially responded to ICIs, later relapsed and acquired therapeutic resistance [117]. The role of miRNAs as predictors of response to ICIs has not been elucidated yet. However, several studies have explored how miRNAs regulate the immune checkpoint signaling pathways, leading the way for the discovery of new predictive biomarkers.

Helicobacter Pylori, which is the most common cause of GC, promotes PD-L1 expression and causes an immune escape by downregulating miR-200b and miR-152. Furthermore, a single nucleotide mutation in 3 -UTR of PD-L1 leads to protein overexpression by disrupting the complementarity between miR-570 and its 3 -UTR binding site. This mutation is linked with high PD-L1 levels in GCs [118]. Moreover, according to the findings of a large population-based study from The Cancer Genome Atlas (TGCA) project, a cluster of EBV-miRNAs is linked with a high expression of PD-1/PD-L1 in solid malignancies, including GC [115]. In the context of hematological malignancies, recent studies have found that EBV-associated lymphomas showed high levels of PD-L1. By restoring the expression of PD-L1 targeting tumor suppressor miRNA miR-34a in vitro, PD-L1 expression was reduced and the tumor immunogenicity was increased [119,120]. Thereby, it could be inferred that miR-34a overexpression could be used to subvert PD-L1 induction in

EBV-associated neoplasms. Moreover, miR-34a has been found to be frequently methylated in GIST [121].

In CRC, the tumor suppressor miR-138-5p acts by downregulating PD-L1, leading to cancer cell growth in vitro and tumorigenesis in vivo [122]. In a recent study on a series of CRC samples, low miR-200 and high *ZEB* oncogene expression, which is a profile compatible with EMT, was associated with upregulation of PD-L1 [123]. Moreover, a comprehensive miRNA screening using the TCGA dataset led to the identification of miR-148a-3p as a potential negative regulator of PD-L1 expression, particularly in dMMR/MSI CRC [124].

MiRNAs involved in drug resistance to targeted therapies in GI cancers are summarized in Table 1. The biological materials and molecular techniques used to investigate miRNAs as predictive biomarkers are outlined in Figure 2.

**Figure 2.** Investigational approaches of miRNAs as potential predictive biomarkers. Credits to BioRender.com, accessed date 18 March 2021.

**Table 1.** miRNAs involved in resistance to targeted therapies in GI cancers.

**Table 1.** *Cont.*

carcinoma.

#### **8. Conclusions**

MicroRNAs are well-documented post-transcriptional regulators of gene-expression in physiological and pathological conditions. They are expressed in an organ- and tissuespecific manner and function through different molecular mechanisms. Given that miRNAs can modulate several oncogenic and tumor-suppressing pathways, increasing evidence points out their role as mediators of drug resistance and as therapeutic options. Because miRNAs can be detected rapidly and efficiently in tissues and biofluids, they are the ideal candidate biomarkers to identify patients with primary resistance to a specific targeted therapy and those who have developed acquired resistance. However, regardless of the numerous studies on the matter, only some miRNAs have been extensively proven to act as mediators of resistance to targeted therapies in GI malignancies, by using large patient cohorts for discovery and validation, and in vitro and in vivo modeling for confirmation. In the era of personalized medicine, more investigational studies are needed to translate the use of miRNA to monitor and forecast treatment response and resistance in a clinical setting.

**Author Contributions:** Conceptualization, V.A., F.G., F.L. and M.F.; methodology, V.A., G.B., L.D.S., V.G., L.N. and V.L.; data curation, V.A., F.G. and M.F.; writing—original draft preparation, V.A., F.G., G.B. and L.D.S.; writing—review and editing, E.S., S.R., S.L. and F.L.; supervision, M.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by a grant from the Italian Health Ministry's research program NET-2016–02363853. The funding agency had no role in the design and performance of the study.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data available upon request.

**Conflicts of Interest:** The authors declare no conflict of interest regarding the present work.

#### **References**


**Examining the Relationship between Cost and Quality of Care in the Neonatal Intensive Care Unit and Beyond**

### **Lauren Culbertson, Dmitry Dukhovny and Wannasiri Lapcharoensap \***

Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239, USA;

<EMAIL_ADDRESS>(L.C<EMAIL_ADDRESS>(D.D.)

**\*** Correspondence<EMAIL_ADDRESS>
Received: 30 September 2020; Accepted: 18 November 2020; Published: 19 November 2020

**Abstract:** There is tremendous variation in costs of delivering health care, whether by country, hospital, or patient. However, the questions remain: what costs are reasonable? How does spending affect patient outcomes? We look to explore the relationship between cost and quality of care in adult, pediatric and neonatal literature. Health care stewardship initiatives attempt to address the issue of lowering costs while maintaining the same quality of care; but how do we define and deliver high value care to our patients? Ultimately, these questions remain challenging to tackle due to the heterogeneous definitions of cost and quality. Further standardization of these terms, as well as studying the variations of both costs and quality, may benefit future research on value in health care.

**Keywords:** health care costs; quality; neonatal intensive care; value


*Solvent Effects on Dye Sensitizers Derived from Anthocyanidins for Applications in Photocatalysis DOI: http://dx.doi.org/10.5772/intechopen.87151*

in water purification, the organic dye may be diluted by water or at least erodes or deteriorates from the photocatalyst surface due to continuous interaction with water. Of course, the specific device configuration used during water treatment defines the disposition and interaction of the photocatalyst with the liquid and ultimately defines the severity of the fluid effects on the sensitizer layer. Dye sensitization effect may be a good choice for water treatment, but its effectiveness depends on the device disposition and the sensitizer presentation mainly when it gets in touch with the polluted water.

Among the different natural pigments, anthocyanidins represent an interesting alternative as dye sensitizing naturally obtained pigment. Since dye sensitizer and DSSC advances may be used in solar technology applications such as photocatalysis, this chapter presents interesting information related to anthocyanidins focused on its potential application in renewable energy applications and in particular when used as dye sensitizing pigment in photocatalysis. In particular, the chapter presents information related to an analysis of the effects caused by commonly used solvents to obtain anthocyanidins such as gas phase (as comparative basis), water, ethanol, n-hexane, and methanol and includes discussion on how several electronic properties of interest are subject to different effects in consequence depending on the selected solvent.