Cancer Cell-Derived Granulocyte-Macrophage Colony-Stimulating Factor Is Dispensable for the Progression of 4T1 Murine Breast Cancer

Abstract

We previously reported that 4T1 murine breast cancer cells produce GM-CSF that up-regulates macrophage expression of several cancer promoting genes, including Mcp-1/Ccl2, Ccl17 and Rankl, suggesting a critical role of cancer cell-derived GM-CSF in cancer progression. Here, we attempted to define whether 4T1 cell-derived GM-CSF contributes to the expression of these genes by 4T1tumors, and their subsequent progression. Intraperitoneal injection of anti-GM-CSF neutralizing antibody did not decrease the expression of Mcp-1, Ccl17 or Rankl mRNA by 4T1 tumors. To further examine the role of cancer cell-derived GM-CSF, we generated GM-CSF-deficient 4T1 cells by using the Crisper-Cas9 system. As previously demonstrated, 4T1 cells are a mixture of cells and cloning of cells by itself significantly reduced tumor growth and lung metastasis. By contrast, GM-CSF-deficiency did not affect tumor growth, lung metastasis or the expression of these chemokine and cytokine genes in tumor tissues. By in-situ hybridization, the expression of Mcp-1 mRNA was detected in both F4/80-expressing and non-expressing cells in tumors of GM-CSF-deficient cells. These results indicate that cancer cell-derived GM-CSF is dispensable for the tuning of the 4T1 tumor microenvironment and the production of MCP-1, CCL17 or RANKL in the 4T1 tumor microenvironment is likely regulated by redundant mechanisms.

Keywords: breast cancer; chemokines; cytokines; inflammation; macrophages; tumor microenvironment.

Figure 1

Anti-GM-CSF treatment of 4T1 tumor-bearing mice did not affect tumor weight or the expression of the Mcp-1/Ccl2, Ccl17 or Rankl genes. One hundred μg of either control rat IgG or anti-GM-CSF Ab was intraperitoneally injected on day 0, 3, 7 and 10. Mice were euthanized on day 14. (A) Tumors were harvested from the mice and the weight of each tumor was weighed. (B) The expression of Mcp-1/Ccl2, Ccl17 and Rankle mRNA was evaluated by qRT-PCR. The results are shown as the mean ± SEM. n = 3 for untreated group. n = 5 for IgG- or Ab-treated group.

Figure 2

Generation of GM-CSF-deficient 4T1 cells. (A) The genomic sequence of the targeted region. ATG in green indicates the initiation codon. The PAM sequences are indicated in red. The sequences in blue indicate the sequences for guide RNA. The sequences with underline indicate the primers for PCR. (B) Genomic DNA from each clone was subjected to PCR to detect the presence of indels. (C) The presence of indels was confirmed by DNA sequencing of the PCR products. The presented sequence is the reverse complement of the coding sequence shown in (A). The PAM sequences are indicated in red. Deletions (---) and insertions (in red below the A8-L sequence) were detected within the targeted region. A8-S had a 16-bp insertion containing a part of the original sequence indicated in green. Clone A8 is KO1 in this study. Clone B11 is KO2 in this study but not included in the photo presented as (B).

Figure 3

Characterization of GM-CSF-deficient 4T1 clones. (A) One thousand cells were seeded into a Lab-Tek II chamber slide (8 well) and cultured for 4 days and stained with Diff-Quik (Sysmex, Tokyo, Japan). The magnification of the original photos was 200×. The scale bar indicates 100 μm. (B) One hundred thousand 4T1 parental or clone cells in 2 mL RPMI1640 containing 10% FBS were seeded into 24-well plates and incubated at 37 °C for 3 days. The concentration of GM-CSF in each sample was measured by ELISA. The results are shown as the mean ± SD. n = 3 for each. * p < 0.05, **** p < 0.0001. (C) The induction of Mcp-1 mRNA expression in mouse peritoneal macrophages by the culture supernatants of the parental 4T1 and clones was evaluated by qRT-PCR. LPS (10 ng/mL) was used as positive control. The results are shown as the mean ± SD. n = 3. *** p < 0.001.

Figure 4

The growth of GM-CSF-deficient 4T1 clones in vitro and their progression in vivo. (A) One hundred thousand cells in 4 mL RPMI1640 containing 10% FBS were seeded into 6-well plates and incubated at 37 °C for 4 days and the number of cells was counted. The results are shown as the mean ± SD. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) One hundred thousand cells were injected into the mammary pad of mice and the sizes of tumors were measured and tumor volumes were calculated. Mice were euthanized 4 weeks after the injection and the weight of tumors, the number of lung metastases and the weight of spleens were evaluated. The results are shown as the mean ± SD. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

Expression of Mcp-1, Ccl17 and Rankl mRNA in tumors of GM-CSF deficient 4T1 clones. One hundred thousand cells were injected into the mammary pad of mice. Mice were euthanized 2 weeks after the injection, tumors were harvested from the mice and total RNA was extracted. (A) The expression of each gene was examined by qRT-PCR. (B). The concentration of MCP-1 in sera was measured by ELISA. The results are shown as the mean ± SD. n = 3. ** p < 0.01.

Figure 6

Infiltration of granulocytes, macrophages and Tregs in in tumors of GM-CSF-deficient 4T1 clones. One hundred thousand cells were injected into the mammary pad of mice, and tumors were harvested 2 weeks after the injection. Paraffin sections were prepared and subjected to H&E staining or immunohistochemistry. (A) H & E staining. The magnification of original photos was 200×. The scale bar indicates 100 μm. (B) The numbers of Ly6G-, F4/80- or FoxP3-positive cells in 5 randomly selected 400× fields were counted and the average number per high power field was obtained. The results are shown as the mean ± SD. n = 3.

Figure 6

Infiltration of granulocytes, macrophages and Tregs in in tumors of GM-CSF-deficient 4T1 clones. One hundred thousand cells were injected into the mammary pad of mice, and tumors were harvested 2 weeks after the injection. Paraffin sections were prepared and subjected to H&E staining or immunohistochemistry. (A) H & E staining. The magnification of original photos was 200×. The scale bar indicates 100 μm. (B) The numbers of Ly6G-, F4/80- or FoxP3-positive cells in 5 randomly selected 400× fields were counted and the average number per high power field was obtained. The results are shown as the mean ± SD. n = 3.

Figure 7

Detection of Mcp-1 mRNA in macrophages infiltrating KO4 tumors. Tumors were harvested 2 weeks after the injection of KO4 cells and the expression of Mcp-1 mRNA was examined by in situ hybridization. (A) F4/80 immunohistochemistry. (B) In situ hybridization. Green dots and red dots represent Mcp-1 and Adgre1 (F4/80) mRNA, respectively. Arrows indicate cells with both Mcp-1 and Adgre1 (F4/80) mRNA. The magnification of the original photos was 400×. The scale bar indicates 50 μm.

Figure 8

Correlation between the levels of G-csf (Csf3) mRNA expression by cancer cells and the degree of splenomegaly and tissue congestion. (A) Four 4T1 cell clones (1 × 10⁵ cells in 100 mL) were transplanted into the mammary pad of female BALB/c mice. Four weeks later, mice were euthanized, spleens were harvested from the mice and weights were measured. * p < 0.05. n = 3 for A7, D5 and H1, and n = 2 for A8. (B) The expression level of Csf3 (Gcsf) mRNA by each clone in vitro was evaluated by qRT-PCR. * p < 0.05. n = 3. (C) H&E staining of lung and liver tissue of mice bearing A7 or D5 clone cells. Capillaries and sinuses of lung and liver of D5 tumor-bearing mice were filled with leukocytes. The magnification of the original photos was 400×. The scale bar indicates 50 μm.