# A Criterion of Colorectal Cancer Diagnosis Using Exosome Fluorescence-Lifetime Imaging

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## Abstract

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## 1. Introduction

- Finger rectal examination;
- Irrigoscopy (X-ray examination of the intestine);
- Test for hidden blood in feces;
- Capsule endoscopy (evaluation using an endocapsule with a digital video camera that passes through all parts of the intestine);
- Colonoscopy (endoscopic diagnostic method using a colonoscope with a video camera, if necessary with a biopsy);
- Cancer markers’ blood tests.

## 2. Materials and Methods

^{7}/mL: [16 (8; 20)] for healthy volunteers, [24 (20; 138)] for benign breast diseases patients, [21 (10; 180)] for breast cancer patients, and [22 (13; 154)] for ovarian cancer patients [28]. The variation was not very high. Therefore, these values could be used as a estimation of exosomes concentration in our case.

## 3. Results

- For every exosome in an FLIM image, we calculated the TPAF average lifetime ${\mathrm{t}}_{\mathrm{m}}$. If the ${\mathrm{t}}_{\mathrm{m}}$value fell within the intersection of the intervals corresponding to short and long lifetime interval approximations, then this exosome was ignored. Otherwise, we assigned this exosome the appropriate index: “h”, corresponding to a CP-associated exosome, or “c”, corresponding to a CRC-associated exosome.
- For a whole FLIM dataset corresponding to a definite participant, we calculated the ratio as follows:$${N}_{ch}=\frac{\sum {N}_{c}}{\sum {N}_{h}+\sum {N}_{c}},$$

## 4. Conclusions

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

## References

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**Figure 1.**An illustration of an exosome sample process prepared for MPTflex microscope analysis: photographs of a glass plate with adhesive tape (

**a**), the process of the exosome sample’s positioning on the glass plate (

**b**), and this construction being covered from above with another glass plate (

**c**).

**Figure 2.**Examples of average TPAF lifetime ${\mathrm{t}}_{\mathrm{m}}$images of an exosome sample registered by MPTflex microscope for randomly selected CRC (

**a**) and CP patients (

**b**). The color characterizes ${\mathrm{t}}_{\mathrm{m}}$ values according to the presented legend.

**Figure 3.**The average TPAF lifetime ${\mathrm{t}}_{\mathrm{m}}$ distributions, normalized on an area under the curve, for images of the exosome samples presented in Figure 2. Notations on the legend: CPs—the exosome sample from the randomly selected CRC patient; CRC—the exosome sample from the randomly selected CP patient. The ${\mathrm{t}}_{\mathrm{m}}$ distribution mean values and standard deviations were calculated by ${\mathrm{t}}_{\mathrm{m}}$ value-averaging over images presented in Figure 2a,b.

**Figure 4.**The results of the average TPAF lifetime ${\mathrm{t}}_{\mathrm{m}}$ image of exosome samples processed by a cutting filter: the image for the CRC patient (

**a**) and the image the CP patient (

**b**). The cutting filter had a threshold of 800 photons per pixel. The corresponding unprocessed images are presented in Figure 2.

**Figure 5.**The phasor plots of the average TPAF lifetime ${\mathrm{t}}_{\mathrm{m}}$images of the exosome samples for a CRC patient before (

**a**) and after (

**b**) the cutting filter processing; (

**c**–

**d**)—The same for a CP patient. The cutting filter had a threshold of 800 photons per pixel.

**Figure 6.**The class areas on phasor plots of the average TPAF lifetime ${\mathrm{t}}_{\mathrm{m}}$images of the exosome samples presented in Figure 5: the first class position on the phasor plot for the CRC patient (

**a**) and the CP patient (

**b**), the second class position on the phasor plot for the CRC patient (

**c**) and the CP patient (

**d**). The first class parameters: ${g}_{c,1}=$ 0.80, ${s}_{c,1}=$ 0.14, and ${R}_{1}=$ 0.51; the second class parameters: ${g}_{c,2}=$ 0.53, ${s}_{c,2}=$ 0.23, and ${R}_{2}=$ 0.71. Here, ${g}_{c,j},{s}_{c,j},\mathrm{and}{R}_{j}$ are coordinates of the center and radius of the $j-th$ class.

**Figure 7.**The differentiation of exosomes on the classes described by Formulas (6) and (7). Here, color data points correspond to the exosomes with short average TPAF lifetimes and gray data points correspond to the exosomes with long average TPAF lifetimes. These images are presented unprocessed in Figure 4.

**Figure 8.**The average TPAF lifetime ${\mathrm{t}}_{\mathrm{m}}$ distributions for of the exosome samples, normalized on an area under the curve, for the whole dataset processed by the cutting filter with a threshold of 800 photons per pixel and the circle masks described by Formulas (6) and (7) in a phasor plane. Notations on the legend: CPs—the exosome samples from the whole CRC patient group; CRC—the exosome samples from the whole CP patient group. The ${\mathrm{t}}_{\mathrm{m}}$ distribution mean values and standard deviations were calculated by ${\mathrm{t}}_{\mathrm{m}}$ value-averaging over images presented in Figure 7a,b.

**Figure 9.**Interval approximations of the distribution functions of the short and long average TPAF lifetimes presented in Figure 8. These curves were calculated using Gaussian approximation of the data presented in Table 1. Notations on the legend: CPs—the exosome samples from the whole CRC patient group; CRC—the exosome samples from the whole CP patient group.

**Figure 10.**A box diagram for ${N}_{ch}$values for CP and CRC patient groups, * p-value is $3.35\xb7{10}^{-5}.$ Here, ${N}_{ch}$ is the relative number of CRC-associated exosomes in the FLIM dataset calculated according to Formula (8).

**Table 1.**Mean and standard deviations for the distributions shown in Figure 8.

Mean Value, ns | Standard Deviation, ns | |
---|---|---|

The short average TPAF lifetime distribution | 0.21 | 0.06 |

The long average TPAF lifetime distribution | 0.43 | 0.19 |

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**MDPI and ACS Style**

Borisov, A.V.; Zakharova, O.A.; Samarinova, A.A.; Yunusova, N.V.; Cheremisina, O.V.; Kistenev, Y.V.
A Criterion of Colorectal Cancer Diagnosis Using Exosome Fluorescence-Lifetime Imaging. *Diagnostics* **2022**, *12*, 1792.
https://doi.org/10.3390/diagnostics12081792

**AMA Style**

Borisov AV, Zakharova OA, Samarinova AA, Yunusova NV, Cheremisina OV, Kistenev YV.
A Criterion of Colorectal Cancer Diagnosis Using Exosome Fluorescence-Lifetime Imaging. *Diagnostics*. 2022; 12(8):1792.
https://doi.org/10.3390/diagnostics12081792

**Chicago/Turabian Style**

Borisov, Alexey V., Olga A. Zakharova, Alisa A. Samarinova, Natalia V. Yunusova, Olga V. Cheremisina, and Yury V. Kistenev.
2022. "A Criterion of Colorectal Cancer Diagnosis Using Exosome Fluorescence-Lifetime Imaging" *Diagnostics* 12, no. 8: 1792.
https://doi.org/10.3390/diagnostics12081792