Mesenchymal stem cells govern immune cell maturation in the tumor microenvironment of oral squamous cell carcinoma

doi:10.21203/rs.3.rs-4949024/v1

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Mesenchymal stem cells govern immune cell maturation in the tumor microenvironment of oral squamous cell carcinoma

https://doi.org/10.21203/rs.3.rs-4949024/v1

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Mesenchymal stem cells (MSCs) have a role in the recruitment and modulation of immune cells due to their strong immunomodulatory effects. Due to their immunomodulatory properties, it is undeniable that MSCs could also influence the immune landscape of the tumor microenvironment (TME). Our study traced bone marrow-derived cells using GFP⁺ bone marrow chimeric mice. Compared to the conventional bone marrow transplantation method (BMT), our improved enzyme-cleaved bone marrow harvestation method (cBMT) allows us to collect more bone marrow-derived MSCs than BMT. Using the difference in MSC population in BMT and cBMT, we study the influences of MSCs in the immune environment of oral squamous cell carcinoma (OSCC) tumors. cBMT tumors, which have a higher population of MSCs, created tumors with a histological resemblance to wild-type mice tumors. Interestingly, recruited GFP-positive cells were higher in number in BMT tumors; however, CD45⁺ cells in cBMT tumors were higher. The immune cell landscape in cBMT tumors resembled more closely to that of tumors in normal wild mice. Upon detailed examination, we discovered that mature T and B cells were recruited more into cBMT tumors, while immature macrophages and myeloid-derived suppressor cells invaded more into BMT tumors. Here, we provided insight into how MSCs control the immune landscape in the tumor microenvironment.

Biological sciences/Cancer/Cancer microenvironment

Biological sciences/Cancer/Cancer models

Biological sciences/Cancer/Oral cancer

Biological sciences/Cancer/Tumour immunology

Health sciences/Oncology

Biological sciences/Immunology/Bone marrow transplantation

Biological sciences/Immunology/Tumour immunology

Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into various lineages ¹. MSCs modulate through expression and secretion of growth factors such as vascular endothelial growth factor (VEGF) and stromal-derived growth factor 1 (SDF-1) ^2,3. In addition, MSCs also release secretory factors such as chemokine ligands, which play a role in migrating and recruiting immune cells ^4,5. Due to their multifaceted roles, MSCs have strong immunomodulatory effects. MSCs can activate or suppress the immune system to control the body's homeostasis ⁶. Bone marrow-derived MSCs (BMSCs) are one of the most readily available sources of MSCs. On the other hand, green fluorescent protein (GFP) bone marrow transplantation (BMT) has been widely used to discover the role of bone marrow-derived cells (BMDCs) in various diseases, including cancer ⁷. However, some studies showed the lack of mesenchymal stem cells (MSCs) in conventional bone marrow transplanted mice. Lack of MSCs in the bone marrow transplanted mice drastically affects body homeostasis ^1,8. Our team improved conventional bone marrow transplantation using a novel Collagenese enzyme-cleaved bone marrow transplantation method (cBMT). In this method, we treated our harvested long bone with enzymes to break down the bone marrow cell aggregates before filtering, allowing for breaking large bone marrow aggregates into single cells and passing through the filter. We already discovered that our novel bone marrow transplantation method allowed for harvesting a higher population of LepR⁺ MSCs from the bone marrow, which in turn, better bone healing by recruiting mature osteoblasts^9,10.

MSCs have influence on tumor development through directly promoting the growth of tumor cells, inducing angiogenesis, and increasing tumor-associated immune cell recruitment⁷. By acting as a proinflammatory factor in tumor microenvironment (TME), MSCs, together with cancer cells, recruit inflammatory cells such as monocytes and T lymphocytes ^11,12.

Despite many in-depth studies of the role of MSCs in cancer, the influences of MSCs in the immune microenvironment of oral squamous cell carcinoma (OSCC) have yet to be explored.

In this study, we used the orthotopic murine OSCC models of BMT and cBMT methods to analyze the influences of MSCs on TME immune characteristics. We categorized the immune cells infiltrated into OSCC tumors of BMT and cBMT mice and comparatively studied the differences in immune nature in the absence and presence of MSCs.

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 CD31^13–15. 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	CD3	CD4	CD8	FOXP3	F4/80	CD11b	CD11c	Gr-1	CD20	CD79a	CD138
BMT vs. Wild	▲	▲	▲	▼	▼	▲	▼	▼	▲	▼	▼
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 TME^17,18. 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 cells^22,23. 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.

BMDCs comprise a heterogenous cell population and can differentiated into blood cells, bone, cartilage, fat, and endothelial cells ²⁴. Due to their multilineage differentiation, BMDCs are essential for tumor homeostasis ^16,25. Previous studies showed that cancer cells can actively recruit the BMDCs into TME ^7,16. To effectively trace the BMDC infiltrated into tumors, GFP chimeric mice models are widely used in many studies. GFP chimeric mice are created by lethal radiation followed by tail vein transplantation of GPF-positive bone marrow cells. This method allows researchers to trace BMDCs in various tissues using GFP expression and used in many studies related to bone healing and cancers^9,26. However, some studies have shown controversial results of the GFP transplantation models, such as the interference of BM cell differentiation.

The outcome may be due to insufficient MSC population harvested from GFP transgenic mice since MSCs also aid in BMDC differentiation via chemokine signaling ²⁷. Our previous study discovered only a small population of LepR⁺ stromal cells in the collected bone marrow tissues from the conventional bone marrow transplantation procedure¹⁰. From there, we created the new enzyme-cleaved bone marrow harvesting method, which harvested the higher population of MSCs.

Loss of MSCs leads to loss of bone mass²⁸ and our previous research showed that cBMT mice could repair bone fractures better than BMT mice by promoting maturation of osteoblasts¹⁰. MSCs can aid in the maturation of BMDCs into the osteoblasts and active immune cells.

The histopathological analysis showed that BMT mice contained higher infiltration of immune cell-like cells and GFP⁺ cells in the TME. In contrast, tumor tissues of cBMT and wild mice showed lower infiltration of immune cell-like cells and GFP⁺ cells. GFP⁺ cells in the cBMT and wild mice were found mainly around the tumor periphery, showing the immune-excluded tumor character²⁹. The similarity of histopathological characteristics of the tumor tissues between cBMT and wild-type mice showed that loss of MSCs can change the TME homeostasis. MOC2 tumor has immune-excluded tumor characteristics where the immune-cell infiltration is hindered into the TME ³⁰. These results showed that cBMT tumors are better in preserving immune-excluded tumor characteristics of MOC2 compared to the BMT tumor.

While our study showed the differences in tumor characters histologically, in-depth functional analysis was not performed in this study, such as in-vitro and in-vivo analysis of OSCC cells and MSCs crosstalk. Our study used heterogenic population of bone marrow cells directly harvested from the bone marrow. Although we previously confirmed the higher population of MSCs using cBMT methods, MSC isolation was not performed in this study. Our study focuses on the influences of a heterogenic population of BMDCs with MSCs on the immune microenvironment of OSCC tumors.

On a detailed characterization of infiltrated immune cells in the TME of BMT and cBMT mice, we discovered that immune cell count shift in cBMT compared to the BMT mice was almost identical to the immune cell count shift in wild type mice compared to the BMT mice (see results in Table 1). Although there are still some differences in immune microenvironment between cBMT and wild mice, these results showed that our novel cBMT method is more effective in replicating natural tumor microenvironment and, hence, more suitable for studying cancer and its TME characters than the conventional BMT method.

Mesenchymal stem cells can self-renew and multilineage differentiation ¹. MSCs can be found in various tissues but most abundantly in the adipose tissue and bone marrow. In addition to the multipotency, mesenchymal stem cells are also known to be involved in the modulation of the immune system. MSCs interact with immune cells such as T cells, B cells and macrophages, dendritic cells, and NK cells through their immunoregulatory properties ^4,5. Due to their ability to modulate the immune system, mesenchymal stem cells can change the immune landscape in TME. cBMT method could collect a larger population of MSCs from the bone marrow of GFP mice, allowing us to compare the influence of MSCs on the TME. When MOC2 tumors were transplanted into cBMT and BMT mice, cBMT tumors showed higher infiltration of CD4⁺ T cells and CD8⁺ T cells into the TME. CD4 and CD8 are the widely used mature T cell markers. In addition, CD20⁺ B cells are also increased in the TME of cBMT tumors. The presence of mature T cells and B cells in the TME of cBMT showed that MSCs play roles in the recruitment and maturation of T cells and B cells into the TME.

On the other hand, F4/80⁺ and Gr-1⁺ macrophages become lower in number in the presence of MSCs in TME. Gr-1 is positive in myeloid-derived suppressor cells (MDSCs) and immature macrophages ³¹. Gr-1^hi MDSCs in the lung injury were differentiated from the Gr-1^low MDSCs when BMSCs were administrated to the mice ³². Consistent with our findings, cBMT tumors with a higher MSC population have low infiltration of MDSCs in the TME. These results indicate that MSCs restrict the infiltration of immature monocytes into the TME and potentially aid in the differentiation of immune cells, playing a critical role in tumor development.

In conclusion, this is the pioneer study to compare the infiltrating immune cell population of OSCC TME between conventional bone marrow transplantation and enzymatic bone marrow transplantation methods. Our findings clarified the role of mesenchymal stem cells in immune landscape of TME and potentially provide new insights for using MSCs in cancer therapy.

Mice and cell lines

Female six weeks old wild type mice (C57BL/6J) and eight weeks old GFP transgenic mice (C57BL/6-Tg [CAG-EGFP] OsbC14-Y01-FM131) were purchased from Charles River Laboratories. All mice are kept in a standardized animal facility with a pathogen-free microenvironment. MOC2 cell line (KER-EWL002-FP) was purchased from a Kerafast cell bank. These cells are cultured in the Iscove's Modified Dulbecco's Medium (IMEM)(gibco) made according to the manufacturer's instruction and kept at 37°C in a humidifying incubator with 5% CO₂.

GFP bone marrow chimeric murine models

BMT and cBMT were performed as described previously ¹⁰. BM cells were freshly collected from the femur and tibia bones of GFP transgenic mice. The proximal parts of the bone were cut, and the bone marrow cells were flushed out until the bone tissue turned white. For cBMT, an additional step of treating the flushed-out bone marrow cells in the culture medium with Collagenase type 4 (1mg/mL) and Dispase (2mg/mL) at 37°C for 10 mins was done. The cells were filtered and resuspended within the HBSS solution at the 1.0 x 10⁷ cells/100µL concentration. The BM cells were injected into the lethally irradiated recipient mice, which underwent 10 Gy of whole-body irradiation via the tail vein.

Bone marrow-derived cell primary culture

The collected bone marrow cells were kept in the KBM ADSC-1 solution (Kohjin Bio) at 37°C in a humidifying incubator with 5% CO₂. The cells were allowed to grow until 80% confluency before analyzing.

Immunocytochemistry staining (ICC)

ICC was performed as described by Yoshida et al. ³³. Bone marrow cells were seeded into 6-well plates and waited 24 hours to be seeded. The cells are fixed with 4% paraformaldehyde before washing in 0.5% Tween 20 solution for 5 minutes and 0.05M TBS. The cells were then blocked with Block Ace and incubated with primary antibodies. Primary antibodies used were listed in Supplemental Table S1. TBS wash was performed, followed by secondary antibodies. The secondary antibodies used were listed in Supplemental Table S2. The cells were then washed and stained with nuclear stain DAPI. The staining results are observed using an All-in-One BZ x700 fluorescence microscope.

Chimeric OSCC tumor models

Tumor transplantation was done after two weeks of BMT and cBMT by injecting the MOC2 cells to the right buccal mucosa of GFP chimeric mice at the concentration of 3 x 10⁴ cells/50µL HBSS (Thermo Fisher Scientific). The mice were kept in the pathogen-free microenvironment for three weeks. After three weeks, euthanasia was performed by overdose inhalant anesthesia administration of Isoflurane (Viatris) with oxygen in enclosed chamber until respiratory arrest occurs. Then, the mice were removed from the chamber and cervical dislocation was rapidly performed to assure euthanasia.

Tissue processing for histological examination

The heads bearing the tumor were harvested and fixed in 4% paraformaldehyde solution for 48 hours. The heads were then decalcified in Osteosoft (Sigma Aldrich) at room temperature for 10–14 days. Then, the tissues were dehydrated, starting from 70% ethanol and gradually increasing up to 100% alcohol. Xylene was used as a clearing agent before embedding in the paraffin. The paraffin tissue blocks were cut at 4µm through cross-section and analyzed using HE and IHC staining.

Immunohistochemistry (IHC)

IHC was performed as described by Kanri et al. ³⁴. IHC was done using the antibodies listed in Supplemental Table S1. The tissues were deparaffinized and rehydrated. The tissues were then blocked to inhibit endogenous peroxidase activity. The antigen retrieval was performed according to the manufacturer's instructions. After the antigen retrieval, the sections were blocked with protein block and incubated with the primary antibody at 4°C overnight. The sections were then incubated in the secondary antibody before incubating in the avidin-biotin complex (Vector lab). Diaminobenzidine tetrahydrochloride (DAB) was used for color development. Mayer's hematoxylin (Sigma Aldrich) was used as a counterstained. The staining results were analyzed using an optical microscope (BX53, Olympus).

Double-fluorescent IHC staining

After the antigen retrieval, the tissues were blocked using Block Ace. The primary antibodies were incubated overnight. TBS wash was done, and the secondary antibody incubation was done for 1 hour. Antibodies used were listed in Supplemental Tables S1 and S2. The sections were stained with nuclear stain DAPI. The staining results were analyzed using an All-in-One BZ x700 fluorescence microscope.

Quantification and Statistical Analysis

The sections were analyzed by taking five manually placed photos of x40 magnification. The analysis of the sections was done using Fiji-2 (Version 1.0). For the comparison of two groups, a two-tailed Student's t-test for independent samples with equal variance was used. For the comparison of two or more groups, one-way ANOVA with multiple comparison tests was used. Statistical analyses were performed using Graph-Pad Prism 9.1.1. The p-values less than 0.05 were considered significant. All data were presented as Mean ± SEM.

Ethics approval for animal experiments

All procedures performed in studies involving animals were in accordance with the ethical standards of Okayama University Care and Use of Laboratory Animals guidelines and approved by the Ethics of Animal Experiments committee of Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences (OKU-2020096). We stated that the protocol adheres to the ARRIVE guidelines for reporting animal experiments.

Data availability statement

The datasets in the current study can be made available from the corresponding author upon reasonable request.

Author Contribution

These authors contributed equally: Htoo Shwe Eain and Yamin Soe.HK, HSE, and YS conceptualized and designed the study. HK, HSE, and YS wrote the original draft of the manuscript. HSE, YS, SS and MWO performed bone marrow transplantation. HSE, YS, ZZM, AC and TP performed experiments. HSE and YS performed data analysis. KT, KN, SI, and HN contributed to and oversaw the experimental designs. HK, KT, KN and HN acquired the funding for the research. All authors reviewed and agreed to the published version of the manuscript.

Acknowledgement

We thank teachers, professors, and colleagues from Okayama University, who gave valuable advice and scientific discussions during the process.

Ethics approval for animal experiments

Data availability statement

The datasets in the current study can be made available from the corresponding author upon reasonable request.

Declaration of competing interest

The authors declare that they have no competing interests that could influence the work reported in this paper.

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No competing interests reported.

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Mesenchymal stem cells govern immune cell maturation in the tumor microenvironment of oral squamous cell carcinoma

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Version 1

Abstract

Figures

INTRODUCTION

RESULTS

Enzymatic treatment harvests a higher MSC population from bone marrow tissues

Changes in the MSC population influence the character of tumor tissues

MSCs in TME are involved in the maturation of tumor-infiltrating immune cells in OSCC tumors

DISCUSSION

METHODS

Mice and cell lines

GFP bone marrow chimeric murine models

Bone marrow-derived cell primary culture

Immunocytochemistry staining (ICC)

Chimeric OSCC tumor models

Tissue processing for histological examination

Immunohistochemistry (IHC)

Double-fluorescent IHC staining

Quantification and Statistical Analysis

Ethics approval for animal experiments

Data availability statement

Declarations

Author Contribution

Acknowledgement

Declaration of competing interest

References

Additional Declarations

Supplementary Files

Status:

Version 1