**2. Mesenchymal Stromal**/**Stem Cells and Radiotherapy**

It is generally acknowledged that MSCs can be found ubiquitously in many tissues and are not limited to those of mesodermal origin, such as bone marrow, adipose, muscle and bone.

Previous reports suggested a protective role for MSCs when combined with radiotherapy (RT) [23,24]. In effect, due to their properties, MSCs may be recognized as a therapeutic tool for treating radiation-induced tissue damage [25–27]. Several reports have shown that MSCs skillfully home onto neoplastics tissues [28,29] and together with tissue recovery functions MSCs prepare the microenvironment by controlling inflammatory processes to reduce the inflammation grade [30,31], where they might have the greatest therapeutic impact in vivo [32]. However, the amount of mesenchymal stromal/stem cells that are up-taken into injured tissues may not be sufficient to explain their strong overall protective effect.

The bioactivation of MSCs may be obtained indifferent ways [33] and the molecules secreted by the activated MSCs (MSCs\*) might have an impact on several immune-cell lineages, establishing an advantageous sphere far away from its original location. We have proposed that exosomes liberated from radiation-activated MSCs\* perform important intratumoral and systemic actions [8,12].

We are aware that cellular therapy with MSCs can be problematic in cancer therapy [12]. Therefore, it is important to emphasize that following irradiation MSCs become senescent and the senescence

is associated with production of a senescent-associated secretory phenotype (SASP). The SASP has antitumor activity since it may induce senescence of neighboring cells by paracrine action [34]. Nevertheless, SASP can modify its composition and become much richer in proinflammatory factors and it has become evident that tumor-associated MSCs have a positive effect on tumor growth and the spread of metastasis [35] through the acquisition of a chemo- and radiotherapy resistance mechanisms [36], and it has been suggested that tumor cells can misuse SASP for their own growth [37]. On the other hand, it has been communicated that, in an inflammatory condition, the exosomes contained in the cancer cell secretome might have a role in the change of the normal MSCs cell phenotype toward a malignant one [36], which could be an impediment to MSCs therapeutic use.

Nevertheless, whether this innate tropism of MSCs toward the tumors and metastatic foci is related with cancer promotion or suppression remains controversial [35,37–40], and further studies on the interactions between cancerous cells and stromal components of tumor microenvironments have been proposed, which is imperative to allow the progress of more suitable treatments for cancer.

It is generally accepted that MSCs-based therapies are of major importance in regenerative medicine and, perhaps, in the future a solution for many other medical problems. However, the success of MSCs therapy relies on the efficiency of its administration and the biodistribution, engraftment, differentiation and secreting paracrine factors at the target sites [41]. Until now, there has been no universal delivery route for mesenchymal stromal/stem cells (MSCs) for different diseases [42]. In fact, efficient homing and migration toward lesion sites play an important role and the local transplantation of MSCs in spatial proximity to the lesion, as well as the systemic administration routes are being carefully explored [43]. There is growing evidence that mesenchymal stromal/stem cells based immunosuppression was mainly attributed to the effects of MSCs-derived extracellular vesicles [44] although it seems clear that transplanted MSCs can indeed leave the blood flow and transmigrate through the endothelial barrier, and reach the lesion site [45]. So, both mechanisms must be accepted until the underlying processes are better understood.

We know that in an uninjured mouse, exogenous intravenously injected MSCs rapidly accumulate within the lungs and are cleared from this site to other organs, such as the liver, within days [46]. As far as up-take in the lungs is concerned, the MSCs are able to release a wide variety of soluble mediators, including anti-inflammatory cytokines [47], antimicrobial and angiogenic macromolecules, and exosomes and microvesicles that are secreted to extravascular spaces [48]. All of the above leads us to believe that the amount of MSCs cells that engraft onto injured tissues may not be sufficient to account for their robust overall protective effects.

On the other hand, the induction of the mechanisms of epithelial and endothelial wound healing and the angiogenesis promotion has been attributed to macromolecules included in the exosomes released by the MSCs cells, which act as tools for defending the intestines from the damage produced by necrotizing enterocolitis experimentally induced in animal models [29]. This has been highly promising [23,49], and MSCs may be a well-thought-out therapeutic tool to treat radiation-induced tissue damage [30]. It is essential to highlight that the group of Chapel et al. has started a phase 2 clinical trial (ClinicalTrials.gov Identifier: NCT02814864) for the handling of severe collateral healthy tissue damage after radiation therapy in patients with prostate cancer, and this clinical trial is sustained by numerous reports focused on the use of MSCs for improving the damage severity on normal tissues after radiation treatment [46,50,51]. However, the damage severity and the mechanisms involved in the control of side effects after radiotherapy [52], as well as the role of MSCs in healthy-tissue radio-protection, are quite unknown.

We have included in Figure 1 a graphic summary of the widespread actions done by MSCs and MSCs\*.

*Cells* **2020**, *9*, x FOR PEER REVIEW 4 of 14

**Figure 1.** Graphic and schematic summary of cell actions, tissue response and possible therapeutic application of mesenchymal stromal/stem cells (MSCs) and activated MSCs. **Figure 1.** Graphic and schematic summary of cell actions, tissue response and possible therapeutic application of mesenchymal stromal/stem cells (MSCs) and activated MSCs.

#### **3. Radiation-Activated Mesenchymal Stromal/Stem Cells 3. Radiation-Activated Mesenchymal Stromal**/**Stem Cells**

When we studied the exosome cargo before and after the activation of MSCs with RT, we discovered significant disparities in the results of the proteomic assessment of both samples. We described that there are qualitative, quantitative and functional differences amongst the proteins contained in the exosomes obtained from basal MSCs and activated MSCs\* [8]. For more information in [8] see Supplementary Materials, additional file 1. When we studied the exosome cargo before and after the activation of MSCs with RT, we discovered significant disparities in the results of the proteomic assessment of both samples. We described that there are qualitative, quantitative and functional differences amongst the proteins contained in the exosomes obtained from basal MSCs and activated MSCs\* [8]. For more information in [8] see Supplementary Materials, additional file 1.

These findings demonstrate the profound metabolic change that these activated cell exosomes have undergone and the consequences after activation with radiation. Amongst the proteins representatives in exosomes released from MSCs\*, we highlight the key components of cell–cell or cell–matrix adhesion and include annexin and integrins [8]. Between them, the presence of annexin A1 (ANXA1) is very noteworthy because it is always present in the exosomes released from MSCs\* and constantly absent in MSCs. We verified these results using quantitative mRNA–PCR to measure the mRNA of this protein in MSCs and MSCs\* and confirmed that mRNA is spectacularly induced in MSCs after irradiation [8]. After measuring quantitatively the mRNAs of the proteins of TRAIL, Dkk3 and ANXA1 in umbilical cord stromal stem-cells, before and after cell stimulation with 2 Gy low-energy transfer ionizing radiation, our previously published results [8] show a clear increase in their intracellular levels, compared with the levels found in basal situations (see these results in [8] supplementary material, Figure S2) and notice that the levels of mRNA of TRAIL and Dkk3 at 48 are strongly increased in treated cells compared to the basal levels (*p* < 0.001), whereas the levels of mRNA of ANXA1 are strongly increased at 24 h, and dramatically at 48 h of cell treatment, with the These findings demonstrate the profound metabolic change that these activated cell exosomes have undergone and the consequences after activation with radiation. Amongst the proteins representatives in exosomes released from MSCs\*, we highlight the key components of cell–cell or cell–matrix adhesion and include annexin and integrins [8]. Between them, the presence of annexin A1 (ANXA1) is very noteworthy because it is always present in the exosomes released from MSCs\* and constantly absent in MSCs. We verified these results using quantitative mRNA–PCR to measure the mRNA of this protein in MSCs and MSCs\* and confirmed that mRNA is spectacularly induced in MSCs after irradiation [8]. After measuring quantitatively the mRNAs of the proteins of TRAIL, Dkk3 and ANXA1 in umbilical cord stromal stem-cells, before and after cell stimulation with 2 Gy low-energy transfer ionizing radiation, our previously published results [8] show a clear increase in their intracellular levels, compared with the levels found in basal situations (see these results in [8] supplementary material, Figure S2) and notice that the levels of mRNA of TRAIL and Dkk3 at 48 are strongly increased in treated cells compared to the basal levels (*p* < 0.001), whereas the levels of mRNA of ANXA1 are strongly increased at 24 h, and dramatically at 48 h of cell treatment, with the statistical differences

found 24 and 48 h being very significant (*p* < 0.0001), which supports the massive presence of ANXA1 in the exosomes released by the radiation-stimulated MSCs.
