**3. KLF4-mediated plasticity of MDSCs**

### **3.1 KLF4 promotes cancer development through regulating plasticity of M-MDSCs**

KLF4 is expressed in many tissues and cells types. Besides in epithelial cells, it is also expressed in bone marrow-derived cells and is key to inflammation [52, 53] and monocyte differentiation [54, 55]. However, it was not clear whether and how immune cell-expressing KLF4 is involved in the development of tumor. It is our hypothesis that the overall function of KLF4 depends on its expression in immune cells and in the resident epithelial cells. In the following discussion, we will focus on the role of MDSC-expressing KLF4 in cancer.

To study the function of KLF4 in MDSCs, we used a 4T1 mammary tumor model. This model is unique due to its similar characteristics with human breast cancer, particularly the ability to spontaneously metastasize to lungs. Based on 4T1 cells, we generated stable KLF4 knockdown cells and control cells using siRNA technology. They were designated as siKLF4 and siCon, respectively. We found that in siCon cell-inoculated BALB/c mice tumors were observed as early as Day 9 and the tumor size reached to 18.2 ± 1.6 mm in diameter. However, in siKLF4 cell-inoculated

**113**

α-SMA<sup>+</sup>

EGFP+

**on functional MDSCs**

*KLF4-Mediated Plasticity of Myeloid-Derived Suppressor Cells (MDSCs)*

accumulated in siKLF4 cell-inoculated mice than in siCon group.

although there was no difference in the total number of EGFP<sup>+</sup>

myofibroblasts after they are recruited to the lungs *in vivo*.

KLF4+/+ and KLF4−/<sup>−</sup> group, in KLF4 deficient mice the number of COL1A1<sup>+</sup>

lation that KLF4 regulates the differentiation of M-MDSCs into fibrocytes and

**3.2 KLF4 deficiency compromised cutaneous wound healing depending** 

cells decreased significantly when compared to that in the KLF4+/+ mice. Similarly,

A pressure ulcer (PU) is defined as an injury caused by unrelieved pressure that results in damage to the skin and underlying tissue [59, 60]. They are thought to be caused by local tissue ischemia, interstitial and lymphatic blockage, reperfusion injury,

cells also decreased in KLF4<sup>−</sup>/<sup>−</sup> mice, further supporting our specu-

cells between the

EGFP+

Consistently, in a mouse B16-F10 implantation melanoma model, we showed that KLF4 deficiency in bone marrow drastically reduced lung metastasis accompanied by decreased recruitment of monocytic CCR2+ MDSCs (M-MDSCs) in the lungs. Interestingly, bone marrow KLF4 deficiency was linked with significantly reduced numbers of fibrocytes and myofibroblasts in metastatic lungs [8]. We further performed a cause-effect study to exclude the effect of KLF4-mediated development of MDSCs and to test the direct effect of KLF4-regulated fibrocyte generation from M-MDSCs on tumor metastasis. We sorted M-MDSC subset from the lungs of mice bearing B16-F10 melanoma. They were mixed with B16-F10 tumor cells and then injected wild-type mice with the mixture intravenously. We then induced KLF4 knockout in these mice by tamoxifen injection. In the control mice, they only received B16-F10 tumor cells, but were still injected with tamoxifen or sunflower seed oil as controls. Mice were sacrificed at Day 7 after tumor cell inoculation. We found that no difference was observed in the incidence of lung metastasis between the mice administrated with tamoxifen or sunflower seed oil. However, in the KLF4<sup>−</sup>/<sup>−</sup> and control groups, metastatic nodules in the pulmonary were drastically fewer than those in the KLF4+/+ group. The results strongly suggest that KLF4 controls the process in which M-MDSCs facilitate the seeding and growth of pulmonary metastatic nodules. We also took advantage of the EGFP marker in the transplanted M-MDSCs. We examined MDSC differentiation in the lung by immunofluorescence using COL1A1 and α-SMA antibodies. We found that

mice the primary mammary tumors became visible on Day 14 and the tumor size was only 11.3 ± 1.4 mm in diameter [18]. These data were in agreement with our previous results showing that KLF4 knockdown delayed the onset of mammary tumor development and inhibited lung metastasis in immunocompromised NOD/SCID mice inoculated with MDA-MB-231 human breast cancer cells [56]. We then tested whether MDSCs were involved in KLF4-mediated tumor development. We examined MDSCs in bone marrow, spleen, and tumor by flow cytometry. We found that after implantation of 4 T1 cells, KLF4 knockdown significantly reduced the numbers of MDSCs in bone marrow and spleen when compared to siCon counterparts [18]. As a critical control, we examined the immunosuppressive activities of MDSCs from control cell- and KLF4 knockdown cell-inoculated mice [57, 58]. As expected, MDSCs from siKLF4 cell-inoculated mouse inhibited proliferation of CD4+ and CD8+ T-cell significantly less than their siCon counterparts. The same assay using MDSCs purified from mouse tumors confirmed this observation. Moreover, consistent with higher T cell proliferation upon KLF4 knockdown, the arginase activities in MDSCs from siKLF4 cell-inoculated mice were lower when compared to those in siCon counterparts. Furthermore, we examined the infiltration of T cells into tumor sites by CD3 immunofluorescence staining. We found that there were more T cells

*DOI: http://dx.doi.org/10.5772/intechopen.89151*

#### *KLF4-Mediated Plasticity of Myeloid-Derived Suppressor Cells (MDSCs) DOI: http://dx.doi.org/10.5772/intechopen.89151*

*Cells of the Immune System*

and plastic nature of MDSCs in wound healing.

**3. KLF4-mediated plasticity of MDSCs**

the role of MDSC-expressing KLF4 in cancer.

**of M-MDSCs**

**3.1 KLF4 promotes cancer development through regulating plasticity** 

KLF4 is expressed in many tissues and cells types. Besides in epithelial cells, it is also expressed in bone marrow-derived cells and is key to inflammation [52, 53] and monocyte differentiation [54, 55]. However, it was not clear whether and how immune cell-expressing KLF4 is involved in the development of tumor. It is our hypothesis that the overall function of KLF4 depends on its expression in immune cells and in the resident epithelial cells. In the following discussion, we will focus on

To study the function of KLF4 in MDSCs, we used a 4T1 mammary tumor model. This model is unique due to its similar characteristics with human breast cancer, particularly the ability to spontaneously metastasize to lungs. Based on 4T1 cells, we generated stable KLF4 knockdown cells and control cells using siRNA technology. They were designated as siKLF4 and siCon, respectively. We found that in siCon cell-inoculated BALB/c mice tumors were observed as early as Day 9 and the tumor size reached to 18.2 ± 1.6 mm in diameter. However, in siKLF4 cell-inoculated

can switch phenotypes, display distinct subpopulations, and produce a large variety of cytokines and chemokines [40]. In tissue repair, neutrophils can show their intra-lineage or functional plasticity by pro- or anti-inflammation, during the early stage of a typical wound repair. In addition, in an inflammatory and pro-type 2 microenvironment of a lesion, neutrophils transdifferentiate into antigen presenting cells (APCs) [41]. Such transdifferentiation into APCs has also been studied in rheumatism, where it could drive sustained inflammation, thereby preventing normal repair [42]. Besides neutrophils, macrophages fulfill roles that change over the duration of wound healing [43]. Initially they are bactericidal, and voraciously phagocytose cell and matrix debris, particularly red blood cells and any spent neutrophils at the wound site. These early stage macrophages are called M1 macrophages, and they are pro-inflammatory. Later in the repair process, macrophages develop the pro-repair capacity. These macrophages are called M2 macrophages, and they are anti-inflammatory and pro-reparative. The resting macrophages are called M0 macrophages. Not surprisingly, the plasticity of macrophages, namely the changeable cellular phenotypes and the range of differentiation and activation states, helps to explain the pleiotropic nature of these cells and their complex functions in wound repair [22, 44]. Beside their role in the early inflammatory stage of wound healing, macrophages contribute to tissue remodeling in wound healing by transdifferentiation, notably into endothelial cells [45, 46], a phenotypical plasticity. When compared to those of neutrophils and macrophages, the role of MDSCs and their plasticity in wound healing are less studied [47]. However, there is ample evidence supporting a critical role of MDSC plasticity in repair. For example, as a heterogeneous and immature population of myeloid cells, recruited MDSCs at wound sites can differentiate into macrophages, DCs, and neutrophils [25]. In addition, because of their immunosuppressive function, MDSCs appear to dampen inflammation at the early stage but then promote healing after inflammation wanes by adopting a fate of fibrocytes [11], a cell type that can further differentiate into myofibroblasts that produce extracellular matrix in wound closure [48, 49]. In cancer, a pathological condition considered as "wounds that do not heal," fibrocytes are viewed as a subpopulation of MDSCs [50, 51], further highlighting a dynamic

**112**

mice the primary mammary tumors became visible on Day 14 and the tumor size was only 11.3 ± 1.4 mm in diameter [18]. These data were in agreement with our previous results showing that KLF4 knockdown delayed the onset of mammary tumor development and inhibited lung metastasis in immunocompromised NOD/SCID mice inoculated with MDA-MB-231 human breast cancer cells [56]. We then tested whether MDSCs were involved in KLF4-mediated tumor development. We examined MDSCs in bone marrow, spleen, and tumor by flow cytometry. We found that after implantation of 4 T1 cells, KLF4 knockdown significantly reduced the numbers of MDSCs in bone marrow and spleen when compared to siCon counterparts [18]. As a critical control, we examined the immunosuppressive activities of MDSCs from control cell- and KLF4 knockdown cell-inoculated mice [57, 58]. As expected, MDSCs from siKLF4 cell-inoculated mouse inhibited proliferation of CD4+ and CD8+ T-cell significantly less than their siCon counterparts. The same assay using MDSCs purified from mouse tumors confirmed this observation. Moreover, consistent with higher T cell proliferation upon KLF4 knockdown, the arginase activities in MDSCs from siKLF4 cell-inoculated mice were lower when compared to those in siCon counterparts. Furthermore, we examined the infiltration of T cells into tumor sites by CD3 immunofluorescence staining. We found that there were more T cells accumulated in siKLF4 cell-inoculated mice than in siCon group.

Consistently, in a mouse B16-F10 implantation melanoma model, we showed that KLF4 deficiency in bone marrow drastically reduced lung metastasis accompanied by decreased recruitment of monocytic CCR2+ MDSCs (M-MDSCs) in the lungs. Interestingly, bone marrow KLF4 deficiency was linked with significantly reduced numbers of fibrocytes and myofibroblasts in metastatic lungs [8]. We further performed a cause-effect study to exclude the effect of KLF4-mediated development of MDSCs and to test the direct effect of KLF4-regulated fibrocyte generation from M-MDSCs on tumor metastasis. We sorted M-MDSC subset from the lungs of mice bearing B16-F10 melanoma. They were mixed with B16-F10 tumor cells and then injected wild-type mice with the mixture intravenously. We then induced KLF4 knockout in these mice by tamoxifen injection. In the control mice, they only received B16-F10 tumor cells, but were still injected with tamoxifen or sunflower seed oil as controls. Mice were sacrificed at Day 7 after tumor cell inoculation. We found that no difference was observed in the incidence of lung metastasis between the mice administrated with tamoxifen or sunflower seed oil. However, in the KLF4<sup>−</sup>/<sup>−</sup> and control groups, metastatic nodules in the pulmonary were drastically fewer than those in the KLF4+/+ group. The results strongly suggest that KLF4 controls the process in which M-MDSCs facilitate the seeding and growth of pulmonary metastatic nodules. We also took advantage of the EGFP marker in the transplanted M-MDSCs. We examined MDSC differentiation in the lung by immunofluorescence using COL1A1 and α-SMA antibodies. We found that although there was no difference in the total number of EGFP<sup>+</sup> cells between the KLF4+/+ and KLF4−/<sup>−</sup> group, in KLF4 deficient mice the number of COL1A1<sup>+</sup> EGFP+ cells decreased significantly when compared to that in the KLF4+/+ mice. Similarly, α-SMA<sup>+</sup> EGFP+ cells also decreased in KLF4<sup>−</sup>/<sup>−</sup> mice, further supporting our speculation that KLF4 regulates the differentiation of M-MDSCs into fibrocytes and myofibroblasts after they are recruited to the lungs *in vivo*.

## **3.2 KLF4 deficiency compromised cutaneous wound healing depending on functional MDSCs**

A pressure ulcer (PU) is defined as an injury caused by unrelieved pressure that results in damage to the skin and underlying tissue [59, 60]. They are thought to be caused by local tissue ischemia, interstitial and lymphatic blockage, reperfusion injury, and mechanical deformation of cells by compressive forces [61]. PUs are detrimental to the patients by prolonging their hospital stay, affecting social life-styles, and contributing to negative psychological consequences [62, 63]. Generally, wound healing includes the early inflammatory phase and the later proliferative and remodeling phases [64–66]. However, this process in PU is frequently stalled in the inflammatory stage [67]. This is the reason why PU has been considered a chronic wound [68].

We have reported that KLF4 ablation delayed cutaneous wound healing in KLF4-CreER/KLF4(flox) [69] and RosaCreER/KLF4(flox) double transgenic mice [11], in which KLF4 was knocked out upon tamoxifen induction. To further test the possibility that KLF4 deficiency-induced delay of cutaneous wound healing may be attributed to bone marrow cells, we transplanted bone marrow cells from RosaCreER/KLF4(flox)/β-actin-EGFP triple transgenic mice into wild type C57BL/6 mice and used these chimeric mice to perform full-thickness wound healing experiments. The wound-closure kinetics showed that wound healing was significantly delayed upon KLF4 knockout in bone marrow. In addition, M-MDSCs but not total MDSCs in the skin wounding bed significantly decreased in the KLF4<sup>−</sup>/<sup>−</sup> group compared to those in the KLF4+/+ group. By flow cytometric analysis, after we gated EGFP+ cells and analyzed COL1A1+ CD45+ CD11b+ populations to examine bone marrow-derived fibrocytes in the skin wounding bed, we showed that fibrocytes decreased in KLF4<sup>−</sup>/<sup>−</sup> group compared to those in KLF4+/+ group. This finding was further confirmed by immunofluorescent staining of the wounding bed, as demonstrated by significantly reduced numbers of COL1A1/EGFP and α-SMA/EGFP co-expressing cells in KLF4<sup>−</sup>/<sup>−</sup> group. Moreover, we transplanted bone marrow cells from KLF4/EGFP transgenic mice, in which KLF4-expressing cells are labeled with EGFP [69], to the wild type mice and performed full thickness wound healing experiments. Four days after the wound placement, the wound healing tissues were collected and slides prepared, followed by immunofluorescent staining. We found that KLF4 expressing EGFP cells in the wound bed adapted elongated morphology and were co-localized with those expressing α-SMA, a marker of myofibroblasts that play a critical role in wound healing [70, 71].

KLF4 was highly expressed in M-MDSCs, and we postulated that KLF4 in M-MDSCs may directly regulate the cutaneous wound healing. Because of the highest expression level of FSP-1 in M-MDSCs among all MDSC subpopulations, to test our hypothesis, we used FSP-1-Cre/KLF4(flox) mice to produce PUs [72]. The dorsal skin of WT and FSP-1-Cre/KLF4(flox) (KLF4 null) mice were shaved, gently pulled up and placed between two cylinders of magnets (12 mm in diameter and 5 mm in thickness), producing a compressive pressure of 50 mmHg between the two magnets according to the established PU model [72–74]. A single ischemiareperfusion cycle (I/R) consisted of a period of magnet placement for 16 h followed by a release or rest of 8 h. Three I/R cycles were used in each animal to initiate decubitus ulcer formation. Ulcers were typically formed at Day 3 (at the end of third I/R cycle) accompanied by full-thickness loss of skin. To assess the wound healing of PU, the detached full-thickness skin (ulcered skin) was removed at Day 3 right after the third I/R cycle, and the closure of open ulcer area in each mouse was monitored and photographed consecutively for 10 days. We found that 1 day after the ulcered skin was removed, the opening areas were increased in both WT and KLF4 null mice, probably because of the acute responses. From Day 2 to Day 10, wounds were gradually healed in WT mice, but the healing was delayed in KLF4 null mice as also indicated by an unclosed wound at Day 10. H&E staining showed an increased suprabasal layer of the skin and decreased hair follicle densities. The infiltrated lymphocytes were almost doubled in granule tissue of the skin in KLF4 null mice. These results suggest an elevated inflammatory status in KLF4 null mice. In agreement with reduced numbers of M-MDSCs and fibrocytes upon KLF4 knockout in

**115**

1 × 105

*KLF4-Mediated Plasticity of Myeloid-Derived Suppressor Cells (MDSCs)*

bone marrow in our full-thickness wound healing model, these populations were also decreased in FSP-1-Cre/KLF4(flox) mice in the PU model. Interestingly, we found that the populations of CD11b+Ly6C++ cells, which may represent inflammatory monocytes [75], in both blood and skin wounding beds were increased when compared to those in wild type mice. This observation is consistent with the

MDSC plasticity, and in general, myeloid plasticity, is regulated by the local microenvironment. These cells are environmental sensors and adapters [25]. In tumor, myeloid cells are the most abundant immune cells, and signals within the tumor microenvironment instruct these cells to change their dynamics and plasticity. There are many potential factors/mechanisms in these processes, including hypoxia, tumor ER stress, exosomes, and tumor-derived soluble factors [76]. In the following discussion, we will focus on KLF4-mediated plasticity of MDSCs in

FSP-1, also known as S100A4, is widely accepted as a fibroblast-specific marker [77, 78]. Given the fact that FSP-1 is expressed in more than 90% of monocytes of the host immune system [79] and that it has a "specific" expression in fibroblasts, it is challenging to reconcile the function of FSP-1 at the cellular level between these two very different cell types. On the other hand, fibrocytes are bone marrowderived progenitor cells that can differentiate into myofibroblasts and promote cutaneous would healing and cancer development [20, 51, 80, 81]. Therefore, fibrocytes are very good candidates for carrying the expression/function of FSP-1

It has been reported that fibrocytes can be generated from bone marrow-derived

cells (**Figure 1A**) in the control group.

Ly6GInt subpopulation of MDSCs (P2 in **Figure 1D** and **E**),

cells such as MDSCs [82]. We postulated that KLF4 controls MDSC-mediated generation of fibrocytes. To test this hypothesis and to examine the underlying mechanisms, we isolated spleen cells from KLF4 inducible knockout Rosa26CreER/ KLF4(flox) mice and examined fibrocyte differentiation using an *ex vivo* assay with murine IL-13 and M-CSF [83]. We found that the application of IL-13 and M-CSF

However, the same treatment decreased the number of fibrocytes to 5 ± 2 cells per

known as M-MDSCs [84, 85]. Note that these M-MDSCs had the highest potential for fibrocyte generation (**Figure 1F**), thus supporting the observation that KLF4 deficiency led to significant decrease in FSP-1 expression and fibrocyte generation (**Figure 1A–C**) in the MDSC pool. To test whether KLF4 directly regulates FSP-1 gene expression, we first using two different KLF4 antibodies to perform a chromatin

fen (**Figure 1B**). Furthermore, we examined KLF4 and FSP-1 expression in the process of fibrocyte generation by quantitative RT-PCR analysis. As shown in **Figure 1C**, both KLF4 and FSP-1 mRNA levels were significantly elevated after the application of IL-13 and M-CSF, which was consistent with *ex vivo* generation of fibrocytes. The induction of KLF4 deficiency by 4-OH tamoxifen correlates with a significant decrease in FSP-1 expression, suggesting a KLF4-mediated regulation of FSP-1 in the process. Since splenocytes are a mixed group of cells, we proceeded to examine KLF4 and FSP-1 expression in different subsets of MDSCs from the wild type mouse splenic tissues (**Figure 1D**). Highest levels of KLF4, FSP-1, and CCR2 expression

splenocytes when KLF4 was knocked out by induction of 5 μM 4-OH tamoxi-

*DOI: http://dx.doi.org/10.5772/intechopen.89151*

increased inflammation in KLF4 null mice.

**3.3 Mechanisms of KLF4-mediated MDSC plasticity**

cancer and wound healing based on our recent studies.

*3.3.1 KLF4 regulates FSP-1 in fibrocyte generation from MDSCs*

from the host immune cells such as MDSCs to fibroblasts.

resulted in 58 ± 7 fibrocytes per 1 × 105

were found in the CD11b+

#### *KLF4-Mediated Plasticity of Myeloid-Derived Suppressor Cells (MDSCs) DOI: http://dx.doi.org/10.5772/intechopen.89151*

*Cells of the Immune System*

and mechanical deformation of cells by compressive forces [61]. PUs are detrimental to the patients by prolonging their hospital stay, affecting social life-styles, and contributing to negative psychological consequences [62, 63]. Generally, wound healing includes the early inflammatory phase and the later proliferative and remodeling phases [64–66]. However, this process in PU is frequently stalled in the inflammatory stage [67]. This is the reason why PU has been considered a chronic wound [68]. We have reported that KLF4 ablation delayed cutaneous wound healing in KLF4-CreER/KLF4(flox) [69] and RosaCreER/KLF4(flox) double transgenic mice [11], in which KLF4 was knocked out upon tamoxifen induction. To further test the possibility that KLF4 deficiency-induced delay of cutaneous wound healing may be attributed to bone marrow cells, we transplanted bone marrow cells from RosaCreER/KLF4(flox)/β-actin-EGFP triple transgenic mice into wild type C57BL/6 mice and used these chimeric mice to perform full-thickness wound healing experiments. The wound-closure kinetics showed that wound healing was significantly delayed upon KLF4 knockout in bone marrow. In addition, M-MDSCs but not total MDSCs in the skin wounding bed significantly decreased in the KLF4<sup>−</sup>/<sup>−</sup> group compared to those in the KLF4+/+ group. By flow cytometric analy-

to examine bone marrow-derived fibrocytes in the skin wounding bed, we showed that fibrocytes decreased in KLF4<sup>−</sup>/<sup>−</sup> group compared to those in KLF4+/+ group. This finding was further confirmed by immunofluorescent staining of the wounding bed, as demonstrated by significantly reduced numbers of COL1A1/EGFP and α-SMA/EGFP co-expressing cells in KLF4<sup>−</sup>/<sup>−</sup> group. Moreover, we transplanted bone marrow cells from KLF4/EGFP transgenic mice, in which KLF4-expressing cells are labeled with EGFP [69], to the wild type mice and performed full thickness wound healing experiments. Four days after the wound placement, the wound healing tissues were collected and slides prepared, followed by immunofluorescent staining. We found that KLF4 expressing EGFP cells in the wound bed adapted elongated morphology and were co-localized with those expressing α-SMA, a marker of myofibroblasts that play a critical role in wound healing [70, 71]. KLF4 was highly expressed in M-MDSCs, and we postulated that KLF4 in M-MDSCs may directly regulate the cutaneous wound healing. Because of the highest expression level of FSP-1 in M-MDSCs among all MDSC subpopulations, to test our hypothesis, we used FSP-1-Cre/KLF4(flox) mice to produce PUs [72]. The dorsal skin of WT and FSP-1-Cre/KLF4(flox) (KLF4 null) mice were shaved, gently pulled up and placed between two cylinders of magnets (12 mm in diameter and 5 mm in thickness), producing a compressive pressure of 50 mmHg between the two magnets according to the established PU model [72–74]. A single ischemiareperfusion cycle (I/R) consisted of a period of magnet placement for 16 h followed by a release or rest of 8 h. Three I/R cycles were used in each animal to initiate decubitus ulcer formation. Ulcers were typically formed at Day 3 (at the end of third I/R cycle) accompanied by full-thickness loss of skin. To assess the wound healing of PU, the detached full-thickness skin (ulcered skin) was removed at Day 3 right after the third I/R cycle, and the closure of open ulcer area in each mouse was monitored and photographed consecutively for 10 days. We found that 1 day after the ulcered skin was removed, the opening areas were increased in both WT and KLF4 null mice, probably because of the acute responses. From Day 2 to Day 10, wounds were gradually healed in WT mice, but the healing was delayed in KLF4 null mice as also indicated by an unclosed wound at Day 10. H&E staining showed an increased suprabasal layer of the skin and decreased hair follicle densities. The infiltrated lymphocytes were almost doubled in granule tissue of the skin in KLF4 null mice. These results suggest an elevated inflammatory status in KLF4 null mice. In agreement with reduced numbers of M-MDSCs and fibrocytes upon KLF4 knockout in

CD45+

CD11b+

populations

sis, after we gated EGFP+ cells and analyzed COL1A1+

**114**

bone marrow in our full-thickness wound healing model, these populations were also decreased in FSP-1-Cre/KLF4(flox) mice in the PU model. Interestingly, we found that the populations of CD11b+Ly6C++ cells, which may represent inflammatory monocytes [75], in both blood and skin wounding beds were increased when compared to those in wild type mice. This observation is consistent with the increased inflammation in KLF4 null mice.
