Chemotherapy-generated cell debris stimulates colon carcinoma tumor growth via osteopontin

Abstract

Colon cancer recurrence after therapy, such as 5-fluorouracil (5-FU), remains a challenge in the clinical setting. Chemotherapy reduces tumor burden by inducing cell death; however, the resulting dead tumor cells, or debris, may paradoxically stimulate angiogenesis, inflammation, and tumor growth. Here, we demonstrate that 5-FU-generated colon carcinoma debris stimulates the growth of a subthreshold inoculum of living tumor cells in subcutaneous and orthotopic models. Debris triggered the release of osteopontin (OPN) by tumor cells and host macrophages. Both coinjection of debris and systemic treatment with 5-FU increased plasma OPN levels in tumor-bearing mice. RNA expression levels of secreted phosphoprotein 1, the gene that encodes OPN, correlate with poor prognosis in patients with colorectal cancer and are elevated in chemotherapy-treated patients who experience tumor recurrence vs. no recurrence. Pharmacologic and genetic ablation of OPN inhibited debris-stimulated tumor growth. Systemic treatment with a combination of a neutralizing OPN antibody and 5-FU dramatically inhibited tumor growth. These results demonstrate a novel mechanism of tumor progression mediated by OPN released in response to chemotherapy-generated tumor cell debris. Neutralization of debris-stimulated OPN represents a potential therapeutic strategy to overcome the inherent limitation of cytotoxic therapies as a result of the generation of cell debris.-Chang, J., Bhasin, S. S., Bielenberg, D. R., Sukhatme, V. P., Bhasin, M., Huang, S., Kieran, M. W., Panigrahy, D. Chemotherapy-generated cell debris stimulates colon carcinoma tumor growth via osteopontin.

Keywords: angiogenesis; cancer; macrophage.

Figure 1

5-FU–generated colon carcinoma cell debris stimulates tumor growth. A) CT26 tumors (5 × 10⁵ living cells) treated systemically with 5-FU (30 mg/kg every 3 d). Treatment was initiated once tumors reached 100–200 mm³ (n = 5 mice/group). B) CT26 tumors (1 × 10⁴ living cells) treated systemically with 5-FU (30 mg/kg every 3 d) starting on the day of injection (n = 5 mice/group). C–E) Flow cytometry analysis of apoptotic (annexin V positive/PI negative; bottom right quadrant), necrotic (annexin V negative/PI positive; upper left quadrant), and late apoptotic/necrotic (annexin V positive/PI positive; upper right quadrant) cell death via annexin V/PI staining of whole population in vitro cell cultures of CT26 (C), MC38 (D), and RKO (E) cells that were treated with 5 µM 5-FU for 72 h vs. control (n = 3/group). F, G) Debris-stimulated CT26 (F) and MC38 (G) tumor growth from 5-FU–generated dead cells coinjected with a subthreshold inoculum of 1 × 10⁴ living cells in Balb/c and C57BL/6 mice, respectively (n = 5–10 mice/group). H) Debris-stimulated RKO tumor growth from 5-FU–generated dead cells coinjected with a subthreshold inoculum of 2 × 10⁵ living cells in SCID mice (n = 5 mice/group). I) 5-FU–generated CT26 dead cells (9 × 10⁵) coinjected with low inoculums of CT26 (1 × 10² or 1 × 10³ living cells; n = 5 mice/group). Data are presented as means ± sem. Two-tailed Student’s t test for final tumor measurements were used throughout unless specified; 1-way ANOVA analysis was used for comparison of 3 or more groups. **P < 0.01, ***P < 0.001.

Figure 2

5-FU–generated colon tumor cell debris stimulates OPN secretion by macrophages and tumor cells. A) Angiogenic cytokine/chemokine profile of CM from RAW264.7 macrophages exposed to 5-FU–generated CT26 dead cells compared with macrophages or dead cells alone. B) ELISA quantification of OPN concentration in CM from RAW264.7 macrophages exposed to 5 µM 5-FU (1 h) and 5-FU–generated CT26 dead cells (1 h) compared with control macrophages or dead cells alone (n = 3/group). C, D) ELISA quantification of murine OPN released by primary resident peritoneal macrophages exposed to 5-FU–generated CT26 (C) or MC38 (D) dead cells vs. macrophages or dead cells alone; macrophages isolated from Balb/c or C57BL/6 mice, respectively (n = 3/group). E) ELISA quantification of human OPN released from primary human monocyte-derived macrophages exposed to 5-FU–generated RKO dead cells vs. macrophages or dead cells alone (n = 3/group). F) ELISA quantification of murine OPN (left) and human OPN (right) in plasma from SCID mice injected with debris-stimulated tumors (9 × 10⁵ 5-FU–generated RKO dead cells alone, 2 × 10⁵ living RKO alone, or 2 × 10⁵ living RKO coinjected with 9 × 10⁵ 5-FU–genererated RKO dead cells; n = 4–5/group). G) Viability of MS1 mouse ECs treated with CM from RAW264.7 macrophages pretreated with control, 3 µg IgG/ml, or 3 µg anti-OPN Ab/ml and exposed to 5-FU–generated CT26 (left) or MC38 (right) dead cells (n = 12/group). Data are presented as means ± sem. Two-tailed Student’s t test was used throughout unless specified. **P < 0.01, ***P < 0.001.

Figure 3

Elevated SPP1 gene expression levels are associated with poor prognosis and tumor recurrence in patients with colorectal cancer. A) K-M analysis exhibiting the correlation between SPP1 RNA expression and survival probability of patients with colon adenocarcinoma (n = 283 patients). P = 0.017. B) K-M analysis exhibiting the correlation between SPP1 gene expression and survival probability of patients with rectum adenocarcinoma (n = 94 patients). P = 0.016. C) Gene Expression Omnibus data analysis of SPP1 gene expression in patients with colorectal cancer who have received FOLFOX chemotherapy comparing patients with recurrence vs. no recurrence (n = 166 patients). P = 0.03. We used the log-rank test to evaluate the significance of K-M curves. P values were calculated in R using Limma for moderate T statistics between the 2 groups. *P < 0.05.

Figure 4

Debris-stimulated tumor growth is OPN dependent. A) Debris-stimulated MC38 (1 × 10⁴ living MC38 coinjected with 9 × 10⁵ 5-FU–generated MC38 tumor cell debris) tumor growth in OPN KO mice compared with WT mice (n = 4–5 mice/group). B) ELISA quantification of OPN in plasma from WT and OPN KO mice that were injected with debris-stimulated MC38 tumors or control non-tumor–bearing WT and OPN KO mice (n = 4–5/group). C) Debris-stimulated CT26 tumor growth (1 × 10⁴ living CT26 coinjected with 9 × 10⁵ CT26 tumor cell debris) treated with anti-OPN Ab or control IgG (20 µg Ab/mouse every 3 d; n = 5 mice/group). D) ELISA quantification of OPN in plasma from mice that were injected with debris-stimulated CT26 tumors treated with control IgG or anti-OPN Ab (n = 5 mice/group). E) CT26 tumors treated with 5-FU (30 mg/kg every 3 d) and/or anti-OPN Ab (20 µg/mouse every 3 d; n = 5 mice/group). Data are presented as means ± sem. Two-tailed Student’s t test for final tumor measurements were used throughout unless specified; 1-way ANOVA analysis was used for comparison of 3 or more groups. **P < 0.01, ***P < 0.001.