Interactions between colon cancer cells and tumor-infiltrated macrophages depending on cancer cell-derived colony stimulating factor 1

Figures & data

Figure 1. The CD68 and CSF1 expression in colon cancer tissue. (A) Numbers of CD68⁺ cells in normal mucosa, peritumoral or intratumoral stoma of 46 cases of primary colon cancer. Each symbol represented one tissue sample. (B) The correlation of infiltrated CD68⁺ cell number between normal tissue and intratumoral stroma (upper) and peritumoral and intratumoral stroma (down). (C) (Upper) A typical view of CSF1 expression in peritumoral and tumor tissue in colon cancer (small: 100×; large: 400×). (Down) The percentage of CSF1-high cases in normal and tumor samples. (D) A typical CD68 and CSF1 expression in two consecutive sections (small: 100×; large: 400×). (E) The correlation between CD68⁺ cell numbers and CSF1 expression score in each tumor sample. **p < 0.01

Table 1. 5-y overall survival according to clinicopathological parameters.

Variable	Patients (n)	5-y OS rate (%)	p^a
Gender
Male	45	46.7	0.243
Female	45	44.4
Age
≤60	21	42.9	0.847
60˜70	31	61.3
>70	38	34.2
Location
Ascending colon	36	38.9	0.250
Transverse colon	5	20.0
Descending colon	49	53.1
Tumor (T)
1–2	6	100.0	0.300
3	70	44.3
4	14	35.7
Lymph node (N)
0	55	52.7	0.003
1	26	38.5
2	9	22.2
Metastasis (M)
0	89	46.1	0.505
1	1	0.0
Stage
I–II	55	52.7	0.002
III–IV	35	34.3
Grade
Poor	3	66.7	< 0.001
Poor-moderate	73	46.6
Moderate-well	11	45.5
Well	3	0.0
Tumor type
Non-mucinous	78	47.4	0.190
Mucinous	12	33.3
Growth pattern
Infiltrating	72	43.1	0.042
Pushing	18	55.6

Abbreviations: OS, overall survival; a: log-rank p value.

Figure 2. Survival of colon cancer patients categorized by CSF1 and CD68 expression. (A) All colon cancer cases in tissue microarray with follow-up data (n=90) were categorized according to expression of CSF1 (left) and CD68 (right) respectively, and survival analysis between two subgroups was performed. (B) Survival analysis was performed between subgroups based on a prognostic score combined with CSF1 and CD68. Low: CSF1_loCD68_lo; Intermediate: CSF_loCD68_hi/CSF_hiCD68_lo; High: CSF1_hiCD68_hi. Survival curves were plotted by Kaplan–Meier method and the log-rank test was used to determine statistical significance.

Table 2. Cox proportional hazard model in 90 colon cancer cases.

Variable	Relative risk	95% CI	p
Stage
I–II	0.522	0.268–1.015	0.055
III–IV	1.000
Grade
Poor	0.209	0.019–2.350	0.205
Poor-moderate	0.162	0.042–0.619	0.008
Moderate-well	0.291	0.064–1.324	0.110
Well	1.000
Growth pattern
Infiltrating	2.496	0.873–7.136	0.088
Pushing	1.000
Prognostic score
Low	3.603	1.289–10.071	0.015
Intermediate	3.114	1.153–8.412	0.025
High	1.000

Abbreviations: CI, confidence interval.

Table 3. Intratumoral CSF1 and CD68 expression in relation to clinicopathological parameters of colon cancer patients.

	CSF1 expression			CD68 expression
Variable	Low	High	p	Low	High	p
Gender
Male	32	39	0.605	38	33	0.078
Female	26	39		45	20
Age
≤60	25	25	0.130	34	16	0.440
60–70	13	30		25	18
>70	20	23		24	19
Location
Ascending colon	19	27	0.962	25	21	0.329
Transverse colon	4	6		8	2
Descending colon	35	45		50	30
Tumor (T)
1–2	6	10	0.669	5	11	0.036
3	46	58		67	37
4	5	10		10	5
Lymph node (N)
0	26	52	0.027	43	35	0.230
1	23	16		26	13
2	9	10		14	5
Metastasis (M)
0	54	76	0.401	77	53	0.081
1	4	2		6	0
Stage
I–II	26	51	0.020	41	36	0.027
III–IV	32	27		42	17
Grade
Poor	9	6	0.523	6	7	0.298
Poor-moderate	38	56		59	35
Moderate-well	6	10		7	9
Well	5	6		8	3
Tumor type
Non-mucinous	51	70	0.739	73	48	0.635
Mucinous	7	8		10	5
Growth pattern
Infiltrating	48	63	0.767	68	43	0.907
Pushing	10	15		15	10

Figure 3. Co-culture with THP-1-derived macrophages promoted CSF1 production in HT-29 colon cancer cells. (A) A diagrammatic illustration for the co-culture procedure. Upper: To obtain the co-culture medium, 2×10⁵ of HT-29 cells were co-cultured with intended numbers of THP-1-derived macrophages for 24 h, then the co-culture medium and cells were collected and measured separately. Down: To obtain conditioned medium (CM), HT-29 cells and THP-1-derived macrophages were cultured alone in new medium for another 24 h after co-culture, then the CM were collected. (B) CSF1 concentration in THP-1-derived macrophages, HT-29 cells and co-culture medium for 12 h and 24 h. Mφ, the CM of THP-1-derived macrophages alone; HT-29, the CM of HT-29 cells alone; Mφ+HT-29, the co-culture medium of THP-1-derived macrophages and HT-29 cells. (C) CSF1 transcription in THP-1-derived macrophages and HT-29 cells after intended period of co-culture. (D) CSF1 concentration in CM of THP-1-derived macrophages and HT-29 cells, alone or 24 h after the co-culture. (E) CSF1 transcription in THP-1-derived macrophages and HT-29 cells co-cultured in different cell ratio. All subgroups were compared to the control. (F) A typical immunofluorescent image for CSF1 expression pattern in HT-29 cells, alone or after 24 h of co-culture with macrophages. Mφ represented THP-1-derived macrophages. The RT-PCR data were normalized to the control and shown as fold change. Each bar represents the mean±SD (n = 3, *p < 0.05, **p < 0.01).

Figure 4. IL-8 promoted CSF1 in HT-29 cells via PKC pathway. (A and B) IL-8 transcription (A) and concentration in CM (B) of THP-1-derived macrophages and HT-29 cells, alone or after co-culture. (C) THP-1-derived macrophages and HT-29 cells were cultured alone or co-cultured with/without IL-8 NAb (2 μg/mL) for 24 h. Then cells were collected respectively and CSF1 transcription was measured. (D) HT-29 cells were co-cultured with THP-1-derived macrophages with/without IL-8 NAb (2 μg/mL) for 24 h, then were cultured alone in new medium for another 24 h. CSF1 concentration in HT-29 CM was measured. (E) HT-29 cells were treated by phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) for 24 h, and CSF1 transcription was measured. (F) HT-29 cells co-cultured with THP-1-derived macrophages with/without IL-8 NAb were measured for phosphorylation of PKC isoforms. Mφ represented THP-1-derived macrophages. The qRT-PCR data were normalized to the control and shown as fold change. Each bar represents the mean ± SD (n = 3, *p < 0.05, **p< 0.01).

Figure 5. HT-29-derived CSF1 recruited THP-1-derived macrophages and directed their cytokines/chemokines production. (A) The CM of THP-1-derived macrophages and HT-29 cells were applied to Transwell assay, and numbers of THP-1-derived macrophages recruited by CM were counted. The SW480 CM was used as a negative control for cancer cell-derived CSF1. (B) The HT-29 CM, alone or co-cultured, were applied for Transwell assay, with/without CSF1 NAb (100 ng/mL), and recruited THP-1-derived macrophages were counted. (C) The CM of HT-29 transfected by control or CSF-shRNA were used for Transwell assay. The recruited THP-1-derived macrophages were counted. (D) THP-1-derived macrophages were co-cultured with HT-29 cells transfected by control or CSF1-shRNA for 24 h, and the transcription of typical pro- or anti-inflammatory cytokines/chemokines was measured. The cytokines/chemokines with significant change were exhibited. Mφ-HT-29: HT-29 CM after 24 h of co-culture; Mφ-SW480: SW480 CM after 24 h of co-culture. The qRT-PCR data were normalized to the control and shown as fold change. Each bar represents the mean ± SD (n = 3, *p < 0.05, **p< 0.01).

Figure 6. Adenovirus-mediated intratumoral CSF1 over-expression affected macrophages infiltration and tumor growth in vivo. (A) A diagrammatic illustration for the tumor model study procedure. 1 × 10⁷ HT-29 cells in 100 μL RPMI 1640 medium was subcutaneously injected into bilateral legs of BALB/c nude mice. After 12 d of tumor inoculation, recombinant adenovirus vector expressing human CSF1 (Ad-hCSF1) or control vector (both 5 × 10¹⁰ vp/100 μL) was injected into the right tumor. Mice were sacrificed and tumors were isolated on day 27. (B) (Left) Typical views of CD68⁺ cells in tumor xenografts treated with control vector or Ad-hCSF1 (400×). (Right) The mean number of CD68⁺ cells infiltrated in tumor. (C) mRNA levels of human CSF1 (upper) and mouse IL-8 (down) in right tumors of two groups were analyzed after 15 d of adenovirus vector injection. (D) Tumor volume in two groups was measured every 3 d and statistical analysis was performed. Tumor volumes were calculated as (length × width²)/2. (E) Mean colon cancer xenograft weights on day 27, treated with control vector or Ad-hCSF1. Data were represented as mean ± SD (n = 5, *p < 0.05, ** p < 0.01).

Figure 7. The schematic diagram depicting interactions between colon cancer cells and tumor infiltrated macrophages depending on CSF1.