The differentiation of hematopoietic stem cells was reprogrammed in advanced tumor-bearing mice

: Background: Hematopoiesis and the differentiation of HSCs have been proved to not only play important roles in cancer progression but also be changed or reprogrammed by the tumor microenvironment itself. In this study, we investigated the changes of HSCs differentiation in advanced tumor-bearing mice. Methods: The tumor-bearing mice model was established by subcutaneously inoculating with xenografts of B16-F10 mouse melanoma cells into the right back of male wild-type C57BL/6 mice. Hematopoietic stem cells and multilineage differentiation were evaluated using blood routine, HE-staining, flow cytometry assay and HSCs culture techniques. Results: The multilineage differentiation of hematopoietic stem cells was reprogrammed in vivo . Especially, the differentiations of megakaryocyte and erythrocyte were blocked, while myeloid cell and lymphoid cell differentiation was encouraged in advanced tumor-bearing mice. Conclusion: In this study we showed the potential mechanism of hematopoietic disorder in tumor condition from a respective of hematopoietic stem cell and multilineage differentiation, which provided new knowledge regarding cachexia.


Antibody staining and flow cytometry
Bone marrow cells (BMCs), spleen cell (SPCs), thymic cells (THCs) and peripheral blood (PBCs) were isolated freshly. Then cells were strained through a 40-um strainer in the presence of phosphate-buffered saline and red blood cells were removed. Cells were stained with antibody for 20 minutes at 4°C. Then cells were subjected to flow cytometry analysis.

HSCs culture techniques
MethoCult™ GF M3434, purchased from STEMCELL, is optimized for the growth and enumeration of hematopoietic progenitor cells in colony-forming unit (CFU) assays of mouse bone marrow cells.

Statistical analysis
Data were expressed as means ± the standard errors of means. Two-tailed Student's t-test was used to analyze the difference between 2 groups. The data were analyzed using SPSS ver. 19 (IBM Corp., Armonk, NY, USA). P<0.05 was considered statistically significant.

Advanced tumor-bearing mice had loss of weight, heavier spleen.
The tumor-bearing mice model was established by subcutaneously inoculating with xenografts of B16-F10 mouse melanoma cells. After 3 weeks, the conditions of mice were evaluated. As shown in Figure 1A, Tumor-bearing mice had loss of weight.
The HE results of femur between control and test group had no significant difference ( Fig 1B). But the structure of tumor-bearing mice' spleen was totally damaged. In addition, the spleen of tumor-bearing mice was heavier than normal mice spleen ( Fig   1C). Advanced tumor-bearing mice had severe hematopoietic abnormalities.
To found out the changes of hematopoietic system in the advanced tumor condition, we did blood routine and found there were severe anomalies of the hematopoietic system in the tumor-bearing mice compared to normal mice. Consistent with the clinic symptoms of cachexia, advanced tumor-bearing mice has severe anemia, thrombocytopenia, granulocytosis and mononucleosis (Fig 2 and Table 1). Gran, granulocytes; Lymph, lymphocytes; Mon, monocytes. Data are presented as mean ± s.d. *, P < 0.05; **, P<0.01.   Advanced tumor-bearing mice had a disrupted HSC repopulation capacity.
Previous studies ascribed severe anemia to the consequence of debilitating chronic disease. But the extensive hematopoietic abnormalities might indicate multilineage disorders in HSCs and/or early progenitors (HPCs). We further assayed the proportion of HSCs/HPCs using flow cytometry ( Fig. 2A). And we found that the proportion of HSCs with Linc-Kit + Sca-1 + phenotype was decreased, while the proportion of HPCs with Linc-Kit + Sca-1phenotype was increased in advanced tumor-bearing mice compared to normal mice (Fig. 2B).  The result of blood routine suggested that advanced tumor-bearing mice also hard granulocytosis. We further examined the changes of myeloid differentiation. The results of flow cytometry showed that the proportion of Gr-1 + CD11b + cells was downregulated in bone marrow, while B220 -CD11b + cells was increased in spleen.
Moreover, consistent with the changes of blood routine, cells with CD11b + phenotype were also increased in PBC. These data suggested that myeloid cell differentiation might be encouraged in advanced tumor-bearing mice.

B-Lymphoid phenotype was induced in advanced tumor-bearing mice.
As showed in blood routine, lymphocytes were also increased in advanced tumor-bearing mice. Then we analyzed the expression of B220 and IgM, which were both important membrane marker in B-lymphoid differentiation process. As showed in Fig. 5A, the proportion of proper-B and mature-B was decreased, while the percentage of immature-B did not much change in BMCs of advanced tumor-bearing mice. Consistent with the bone marrow phenotype, the proportion of mature Blymphocytes with B220 + IgM + phenotype was also decreased in spleen of advanced tumor-bearing mice compared to normal mice. And the proportion of B220 + cells were increased in PBCs of advanced tumor-bearing mice. Taken together, these data revealed that B-Lymphocytes was induced in tumor-bearing mice.  After three weeks the mice model established, the BMCs were isolated freshly and then seed into 6 wells plank. After 10 days, the photos were taken and shown as Figure 7. BFU-E in tumor-bearing mice was smaller than it in normal mice, while CFU-GM and CFU-GEMM were much bigger than that in in normal mice. Those results were consistent with the blood routine and flow cytometry results.

Discussion
At present, when the number of cachectic patients and therefore cancer rates are increasing, it seems particularly appropriate to analyze the different pathophysiologic adaptations that are involved in cancer, with the aim to facilitate the development of a more focused therapeutic approach [21][22][23] . In this research, tumor-bearing mice models were used to extrapolate advanced tumor patients.
After three weeks the tumor-bearing mice model established, we measured the changes of mice's body weight and found that the body weight of tumor bearing mice were much lower than the body weight of normal mice, which proved that the cancer cachexia model was succeed established. Then the hemopoietic system, including bone marrow and spleen, were analyzed by using HE staining. To our surprised, the structure of tumor-bearing mice' spleen was totally damaged. In addition, the spleen of tumor-bearing mice was heavier than normal mice spleen. But the structure of femur between control and test group had no significant difference. The blood routine shown that peripheral blood of tumor-bearing mice had severe hematopoietic abnormalities, severe anemia, thrombocytopenia, granulocytosis.
Previous studies have attributed severe anemia to the consequence of debilitating chronic disease [24,25] . But in this work, we focused on the changes of hematopoiesis in advanced tumor-bearing mice model. By using flow cytometry assay and HSCs culture techniques, we found that tumor-bearing mice had a disrupted HSCs repopulation capacity. The erythrocyte differentiation was blocked in Ery.A, which was responsible for the severe anemia. The decreased CD41+ megakaryocyte contributes to thrombocytopenia observed in the mice bearing the B16-F10 melanoma. As for Myeloid cells, those cells' differentiation was induced in tumorbearing mice, which caused granulocytosis. Another mechanism that is involved in granulocytosis is inflammation. Advanced tumor-bearing mice always have inflammation [26,27] , but in this study, we cannot exclude the role of inflammation in By focusing on the changes of hematopoiesis in advanced tumor-bearing mice model, we found a series of significative evidences. Of course, this study had some shortcomings. Such as that the tumor models grow faster than tumors found in humans; the tumor models are carried out using subcutaneous inoculation and therefore may not reflect the behavior of a tumor in the correct anatomical location.

Conclusion
In conclusion, in this work we try to find new evidences to explain he potential mechanism of hematopoietic disorder in tumor condition from a respective of