Irradiation induces an increase in hematuria from 40 Gy upwards as early as 100 days post-radiation
Hematuria is the main symptom used to measure the evolution of CRC. The criterion of the appearance of hematuria was used to evaluate the kinetics of the evolution of the chronic phase of CRC as a function of the radiation dose. Whole bladder irradiation in a dose range of 20–80 Gy (20, 30, 40, 50, 60, 70 and 80 Gy) was used to determine the optimal dose to induce hematuria.
In order to model the bivariate dynamics (in dose and post-irradiation time) of the evolution of CRC, a flexible nonlinear regression via spline functions was conducted (REFUND package of the R software). The result of this modeling is represented in Fig. 2A as a three-dimensional surface (and its 2D projection as a heat map). On the x-axis is the irradiation dose, on the y-axis the post-irradiation time, and the cumulative dose of erythrocytes per microliter (noted ery/μl) of urine in vertical dimension. In our model, at a dose of 20 Gy, hematuria appears late, beyond 200 days (6 months) after irradiation. At 40 Gy, hematuria appears as early as 100 days post-irradiation (100 ery/μl), indicated by the white arrows. This flexible bivariate modeling thus revealed a significant association between the irradiation dose and the extent of hematuria (p < 0.001). For doses of 60–80 Gy, hematuria also began at 100 days post-irradiation and increased up to 300 days post-irradiation (up to 200 ery/μl). Doses above 40 Gy are too deleterious (weight loss, rat grimace scale) to allow long-term follow-up of the rats. Thus, the results of the data modeling described above suggest quite clearly the 40 Gy dose for this study, as it induces early hematuria without impacting survival beyond 10 months.
Reduction of vascular lesions after MSC treatment
Cystoscopy is used to describe lesions such as telangiectasias, ulcers, papillae and cancerous lesions in patients. It enables discrimination from other etiologies of hematuria, such as renal lesions, primarily. We used cystoscopy in our study model to objectify vascular lesions, to record the number and severity of vascular lesions and to detect any abnormalities (papilla, ulcer).
In Fig. 2B, images of cystoscopies (with lesions circled with a black dotted line) at 6 and 12 months are shown, and in Figs. 2C and D, the number of vascular lesions is shown as a function of the importance of the lesions and the treatment conditions. On the ordinate, the number of vascular lesions is given, and on the abscissa, the different groups as well as the total lesions. A statistical test (ANOVA) was carried out on all the data.
Figure 2C, at 6 months post-irradiation, an absence of vascular lesions was observed for unirradiated and untreated rats. For unirradiated and treated rats, 2 ± 1.1 lesions (Fig. 2C) were observed on average. For untreated irradiated rats, the animals had an average of 4 ± 4.7 lesions. For irradiated and treated rats, on average 5.7 ± 2.9 lesions were observed.
At 12 months post-irradiation, we can observe for unirradiated untreated rats an average of 0.7 ± 0.4 lesions (Figs. 2B, D). For unirradiated untreated rats, no vascular lesions are observed. For the irradiated untreated rats, the animals have an average of 4.3 ± 2.4 lesions. For irradiated and treated rats, an average of 1.3 ± 0.4 lesions is observed.
In conclusion, at 12 months post-irradiation, treatment with MSCs appears to reduce vascular lesions.
The data in Fig. 2E show that irradiation increases the number of micturition, but treatment significantly reduces micturition at 6 months. For unirradiated untreated rats, the number of micturition has an average of 3.5 ± 0.38 micturitions. For the irradiated untreated rats, the animals have an average of 9.75 ± 1.22 micturitions. For irradiated and treated rats, an average of 3.12 ± 0.51 micturitions is observed. Comparing irradiated untreated rats with unirradiated rats at 6 months an increase in micturitions of 2.79 fold is observed (p value < 0.001). Comparing irradiated treated rats with irradiated untreated rats at 6 months, a decrease in micturitions of 3.1 fold is observed (p value < 0.0001). Results are in favor that treatment might restore functionality of bladder after irradiation.
MSCs have a protective effect on the impermeability of the urothelium
MSC treatment restores the structure and functionality of the epithelia after irradiation at the intestinal level [24]. In our study model, we evaluated whether MSC treatment protects the urothelium from irradiation. The function of the uroplakin complex is to ensure the impermeability of the urothelium. A decrease in uroplakin expression is directly related to a loss of urothelial impermeability [28]. Uroplakin III labeling was performed by visible immunohistochemistry. A grading system for uroplakin III expression was set up and measured on the whole urothelium of each animal, as described in the Methods and Materials section. Grade 0 indicates a high level of uroplakin, while Grade 3 indicates no uroplakin (Fig. 3A). The lower grades therefore indicate a urothelium that has retained a normal level of uroplakin III while the higher grades indicate a degraded urothelium. Thus, for each animal the percentage of urothelium in Grades 0–3 was quantified.
A rigorous statistical analysis of the differences in grade percentages cannot therefore be based on standard analysis tools (ANOVA, t tests, etc.) due to the very strong constraints of the input data: the percentages of Grades 0–3 for each animal are not only data between 0 and 1 but have the additional condition of having a sum equal to 1. In order to take this data format into account, a compositional analysis [29] was conducted, which first consisted in transforming the percentage vectors into unconstrained data in order to deploy the classical analysis tools. Due to the presence of zero percentages, a spherical transformation [30] was used.
Figure 3B is a kinetic study of the evolution of the grades comparing 12 months to 8 months for each condition (e.g., 8 months non-irradiated rats compared 12 months non-irradiated rats). Figure 3C shows the difference in uroplakin III expression grades at 12 months for irradiated untreated rats compared to unirradiated rats (Fig. 3C left) and for irradiated untreated rats compared to irradiated treated rats (Fig. 3C right).
When comparing the unirradiated rats at 12 months compared 8 months, there is no change in grades over the course of time (Fig. 3B, unirradiated). When comparing the irradiated untreated rats at 12 months compared 8 months a decrease in Grade 1 by 25% (p value < 0.001), an increase in Grade 2 by 10% (p value = 0.031) and Grade 3 by 15% (p value = 0.001) is observed (Fig. 3B, irradiated untreated). In contrast, the analysis revealed no significant changes in uroplakin III grades in irradiated and treated rats over time (Fig. 3B, irradiated treated). In summary, treatment with MSCs inhibited the effects of irradiation over the course of time (no increase in grades).
Comparing irradiated untreated rats with unirradiated rats at 12 months a decrease in Grade 1 of 27.5% (p value < 0.001), an increase in Grade 2 of 14.25% (p value < 0.001) and Grade 3 of 15.5% (p value = 0.001) is observed (Fig. 3C irradiated untreated compared unirradiated). Irradiation therefore induces the loss of uroplakin III expression after irradiation (Fig. 3C irradiated untreated compared irradiated treated).
Comparing irradiated untreated rats with irradiated treated rats at 12 months shows, for the treated irradiated rats compared to the irradiated rats, a significant decrease in Grade 1 of 13.5% (p value = 0.041), and an increase close to significance in Grades 2 and 3 is observed for the untreated rats (8%, p value = 0.067 and 7.5% p value = 0.082 respectively). Treatment with MSCs thus appears to limit the loss of uroplakin III expression after irradiation.
In conclusion, irradiation increases the loss of uroplakin III expression (Grade 2 and 3). In contrast, MSC treatment limits the loss of uroplakin III expression (Grades 2 and 3) induced by irradiation. MSCs therefore appear to have a protective effect on uroplakin III expression by maintaining its expression over time in treated irradiated rats compared to untreated irradiated rats. These results suggest a protective effect of MSCs on the maintenance of uroplakin III expression and thus a maintenance of urothelial impermeability.
MSCs allow a reduction in irradiation-induced hyperplasia
Hyperplasia is deleterious in the long term as it permanently reduces impermeability and can induce lesions such as metaplasia or precancerous lesions [31]. The aim of the hyperplasia analysis is to assess whether MSC treatment protects the functional integrity of the urothelium as well as preventing regeneration abnormalities. The length of the urothelium with hyperplasia was measured and related to the total size of the urothelium which represents a percentage of the length of the urothelium with hyperplasia, as previously defined [32]. Figure 4A shows representative pictures of the hyperplasia (H) visible on the HES sections. For unirradiated rats (treated or untreated, Fig. 4A, to the left), no hyperplasia was noted in the urothelium (U). In contrast, rats irradiated from 8 months of age showed strong hyperplasia (H) which increased at 12 months (Fig. 4A, irradiated untreated, on the right). When the rats are treated with MSCs, the hyperplasia is more limited and restricted to limited areas (Fig. 4A, irradiated treated, on the right).
As with the uroplakin III grading analyses, compositional analysis based on spherical transformation allows the effect of MSC treatment on urothelial hyperplasia to be assessed (Fig. 4B) as a function of time after irradiation. It appears that treatment with MSCs limits hyperplasia over time compared to the untreated irradiated group. Only the 12-month data are significant and show that the untreated irradiated rats have 16% more urothelium with hyperplasia compared to the MSC-treated irradiated rats (p value = 0.024).
Hyperplasia may be mediated by inflammation. Mast cells are in part responsible for the inflammation in CRC. To investigate whether mast cells could be responsible for the hyperplasia, and whether MSCs could down-regulate this effect, we quantified the number of mast cells (Fig. 5A, B). For irradiated untreated rats, an increase in the number of mast cells (fold change = 1.5 ± 0.56; p value < 0.05) compared with untreated unirradiated rats is observed. For treated irradiated rats, the number of mast cells is not significantly different from the group of irradiated rats. In conclusion, after irradiation there is an increase in mast cells in bladder of untreated irradiated rats. Nevertheless, no observable effect of MSC treatment was observed. This is an indication that inflammation might be related to hyperplasia nevertheless MSC treatment, does not appear to influence inflammation with respect to the number of mast cells.
We retained to go further on the understanding of the mechanistic to support the observations of the integrity of the urothelium and hyperplasia. As shown in Fig. 5C, in the irradiated group of rats, hyperplasia is observed, due to a strong proliferation of basal stem cells without differentiation into superficial cells compared to the treated group. The superficial cells ensure the impermeability of the urothelium through the expression of uroplakin 3. We quantified the number of basal stem cells (labeled with cytokeratin 14) as shown in Fig. 5C. The results in Fig. 5D confirm that irradiation increases the number of basal stem cell, and that treatment is in favor of decreased number of basal stem cells. For irradiated untreated rats, an increase in the number of basal stem cells (fold change = 2.31 ± 0.4; p value < 0.1) compared with untreated unirradiated rats is observed. For treated irradiated rats, the number of basal stem cells is not significantly different from the control group (fold change = 1.46 ± 0.34).
In conclusion, MSCs induce a slight transient hyperplasia at 6 months post-irradiation (not significant) which may be associated with early regeneration of the urothelium. At longer times (8 and 12 months), the group of irradiated rats showed a strong hyperplasia which the MSC treatment seemed to have regulated. This process does not seem to be induced by down regulation of inflammation by MSCs but rather by controlling the regulation of urothelium regeneration. These results therefore suggest that MSC treatment reduces the intensity of hyperplasia during the course of CRC.