Enzymatic treatment harvests a higher MSC population from bone marrow tissues
To collect GFP+ BMDCs, a long bone from the GFP transgenic mice was taken first. The culture medium was used to flush out the bone marrow cells. Wild-type mice were given a lethal dose of radiation to kill the existing bone marrow cells. Then, the collected GFP+ bone marrow cells were injected into the radiated mice via the tail vein, creating chimeric mice with GFP+ bone marrow tissue (Fig. 1A). The additional step of 10 minutes of incubation with the enzyme at 37°C was done for bone marrow harvesting of cBMT (Fig. 1B).
Our previous study confirmed that cBMT method could isolate a higher population of LepR+ bone marrow stromal cells than the conventional BMT method10. When bone marrow tissues are flushed out from the bone marrow, bone marrow cells are attached by the adhesion protein, creating colonies of BM cells. When strained with the single cell filter, these colonies containing many stromal cells are lost in the filter (Fig. 1C). On the other hand, the enzyme treatment to flush out bone marrow tissue can cleave those colonies into single cells, allowing more stromal cells to be collected (Fig. 1E).
To investigate the difference in the MSC population, we cultured the harvested cells from BMT and cBMT methods and study the characters of the cells. We found that the culture cells from the cBMT method have a higher population of spindle-shaped MSCs compared to those from BMT method (Fig. 1D, F).
We immunostained the cells with MSC markers to confirm the presence of MSCs in the cBMT cultures. Since the cell was harvested from a GFP+ transgenic mouse, a GFP marker is used to identify the cell shape. MSCs possess SDF1 and CD105 expression and lack the expression of CD45 and CD3113–15. The bone marrow cells in cBMT cultures showed GFP+SDF1+, GFP+CD105+, GFP+CD45−, and GFP+CD31− cells (Fig. S1A-D).
These results further confirmed the presence of MSCs in cBMT culture.
Combined with our previous finding10, these findings suggest that the cBMT method can harvest the higher MSC population from bone marrow tissues.
Changes in the MSC population influence the character of tumor tissues
MSCs can influence immunomodulation in TME through direct contact or signaling5. On the other hand, BMDCs are essential components in controlling TME16.
To study the influence of the bone marrow-derived mesenchymal stem cells (BMSCs) on the TME of oral squamous cell carcinoma (OSCC), we created the OSCC tumor models in BMT and cBMT chimeric mice. After the bone marrow transplantation was done, the chimeric mice were kept under watch for 14 days to ensure stable BMT transplantation until the cancer was injected. MOC2 tumors were inoculated for 21 days until sacrifice (Fig. 2A).
Next, we investigated the histopathological features of transplanted MOC2 tumors in wild mice, BMT mice, and cBMT mice. Interestingly, increased infiltration of cells with immune cells-like characteristics were found to be infiltrated in the BMT tumors compared to wild tumors and cBMT tumors (Fig. 2B).
To further confirm whether the infiltrated immune cells were derived from bone marrow, we stained the tumor tissues with GFP staining and CD45 staining. Bone marrow-derived GFP+ cells were distributed throughout the tumor tissue in BMT mice, whereas GFP+ cells were found mainly in the periphery of the tumor and had less infiltration into the center of the tumor (Fig. 2C). The number of GFP+ cells infiltrated into the cancer also showed a significant decrease in cBMT tumors compared to BMT tumors (Fig. 2D).
CD45 is the surface marker of immune cells, and the expression level indicates the immune cells in tumor tissues. To analyze the immune cell number in the tumor tissues, we immunostained and counted the CD45+ cells. Interestingly, in contrast to GFP+ cells, CD45+ cells were higher in number in cBMT than in BMT tumors (Fig. 2E, F).
These data suggests that the number of bone marrow-derived cells between the BMT and cBMT tumors differs, and the infiltrated immune cell population in TME may also vary.
cBMT resembles the natural immune landscape while BMT shows a significant disparity of immune cell population
To analyze the immune microenvironments of OSCC tumors in BMT mice, cBMT mice, and wild-type mice, we immunostained and counted the various immune cell markers. First, we compared and studied the change in the immune population of TME. We found that the immune landscape change vastly in BMT tumors compared to tumors of wild mice. CD4+, CD8+, CD11b+, and CD20+ cells were increased in the TME of wild mice tumors compared to that of BMT mice tumors (Fig. 3A). FOXP3+, F4/80+, CD11c+, Gr-1+, CD138+, and CD79a+ cells were reduced in the TME of wild mice tumors compared to that of BMT mice tumors (Fig. 3A). Similarly, we analyzed the immune landscape of the cBMT mice and compared it to that of BMT mice and discovered that cBMT tumors also showed an increase in CD4+, CD8+, CD11b+, and CD20+ cells and a decreased in FOXP3+, F4/80+, CD11c+, Gr-1+, CD138+, and CD79a+ cells.
We then compared the shift in numbers of infiltrated immune cells between BMT and wild tumors, and between BMT and cBMT tumors. We found that the immune cell population in the cBMT tumor shifts similarly to that of wild mice tumor (Table 1).
TABLE 1. Table showing immune cell population change between BMT tumors vs. Wild tumor and BMT tumors vs. cBMT tumors.
Marker
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CD3
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CD4
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CD8
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FOXP3
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F4/80
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CD11b
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CD11c
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Gr-1
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CD20
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CD79a
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CD138
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BMT vs. Wild
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▲
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▲
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▲
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▼
|
▼
|
▲
|
▼
|
▼
|
▲
|
▼
|
▼
|
BMT vs. cBMT
|
▲
|
▲
|
▲
|
▼
|
▼
|
▲
|
▼
|
▼
|
▲
|
▼
|
▼
|
▲ increase compared to BMT tumors ▼ decrease compared to BMT tumors
These results suggest that cBMT can create an immune microenvironment closer to the normal immune system than conventional BMT.
MSCs in TME are involved in the maturation of tumor-infiltrating immune cells in OSCC tumors
For detailed investigation of the influence of MSCs in the immune microenvironment, we analyzed the number of T cells, B cells, and macrophage cells between BMT tumors and cBMT tumors. CD4 and CD8 are the helper T cell and cytotoxic T cell markers, respectively. CD4 and CD8 markers marked the mature T cells in TME17,18. We found that CD4+ helper T cells are increased in the TME of cBMT compared to that of BMT (Fig. 4A, B). Similarly, CD8+ cytotoxic T cells are increased in the TME of cBMT compared to that of BMT (Fig. 4C, D).
CD20 is a pan B cell marker expressed in B lymphocytes before becoming plasma cells19. We discovered that the CD20+ B cell population is higher in the cBMT TME than in BMT TME (Fig. 4E, F).
These results indicate that MSCs are involved in the maturation of T cells and B cells infiltrated into TME.
The F4/80 marker is the pan-marker of macrophages and was positive for both mature and immature macrophages20. F4/80+ cells are reduced in the TME of cBMT tumor compared to that of BMT tumor (Fig. 5A, B). On the other hand, CD11b is expressed in monocytes, granulocytes, and macrophages 21. CD11b+ cell population is slightly higher in cBMT tumors than BMT tumors, although no significant differences exist (Fig. S2A, B). Gr-1 is the marker mainly for the immature myeloid suppressor cells22. Gr-1+ cells are decreased in cBMT tumors compared to BMT tumors (Fig. 5C, D).
Among CD11b+ monocytes, CD11b+Gr-1+ double-positive cells are known to be immature myeloid-derived suppressor cells22,23. Double immunofluorescence showed that BMT tumors has higher CD11b+Gr-1+ cells in the tumor compared to cBMT tumors (Fig. 5E, 5F). These results indicate that presence of MSCs reduced the recruitment of immature macrophages into TME.
All the results showed that MSCs aid in the recruitment of mature BMDC into TME and may involve in maturation of BMDCs in the TME of OSCC tumors.