Discontinuous Schedule of Bevacizumab in Colorectal Cancer Induces Accelerated Tumor Growth and Phenotypic Changes

Abstract

Antiangiogenics administration in colorectal cancer patients seemed promising therapeutic approach. Inspite of early encouraging results, it however gave only modest clinical benefits. When AAG was administered with discontinuous schedule, the disease showed acceleration in certain cases. Though resistance to AAG has been extensively studied, it is not documented for discontinuous schedules. To simulate clinical situations, we subjected a patient-derived CRC subcutaneous xenograft in mice to three different protocols: 1) AAG (bevacizumab) treatment for 30 days (group A) (group B was the control), 2) bevacizumab treatment for 50 days (group C) and bevacizumab for 30 days and 20 without treatment (group D), and 3) bevacizumab treatment for 70 days (group E) and 70 days treatment with a drug-break period between day 30 and 50 (group F). The tumor growth was monitored, and at sacrifice, the vascularity of tumors was measured and the proangiogenic factors quantified. Tumor phenotype was studied by quantifying cancer stem cells. Interrupting bevacizumab during treatment accelerated tumor growth and revascularization. A significant increase of proangiogenic factors was observed when therapy was stopped. On withdrawal of bevacizumab, as also after the drug-break period, the plasmatic VEGF increased significantly. Similarly, a notable increase of CSCs after the withdrawal and drug-break period of bevacizumab was observed (P<.01). The present study indicates that bevacizumab treatment needs to be maintained because discontinuous schedules tend to trigger tumor regrowth, and increase tumor resistance and CSC heterogeneity.

Figure 1

Treatment regimen. The mice were treated with three different regimens to analyze the effect of a discontinuous therapy with bevacizumab. The first regimen (D30) was composed of six mice per group. Group A was treated 30 days with bevacizumab versus the nontreated control, group B. The second regimen (D50) was composed of five mice per group. Group C was treated for 50 days continuously with bevacizumab versus group D treated for 30 days and 20 subsequent days without treatment. The third regimen (D70) was composed of five mice per group. Group E was treated for 70 days continuously with bevacizumab versus group F treated for 70 days with a drug-break period from day 30 to day 50.

Figure 2

Kinetics of tumor growth on colon PDX tumor treated with a discontinuous schedule of bevacizumab. (A) Bevacizumab administered 30 days (group A) induced a significant delay in tumor growth compared to nontreated control (group B). (B) Bevacizumab discontinued at day 30 (group D) induced a rapid resumption of tumor growth as compared to group C. (C) When bevacizumab was stopped at day 30 (group F), there was a slight but notable resumption of tumor growth which was not restored after rechallenge at day 50 compared to group E, treated for 70 days uninterrupted. Data are presented as mean±SEM. .01<*P<.05; .001<**P<.01.

Figure 3

Fluorescence microscopic images of tumors stained for CD31 (green; endothelial cells) and desmin (red; pericytes). The staining with CD31 (A) showed a regression after bevacizumab for 30 days compared to the nontreated tumors; (B) there are no obvious differences in expression of desmin in the two groups. (C) When treatment was maintained for 50 days, the vascularization was still held in check. (D) But when the treatment was stopped, the tumors became highly revascularized by CD31 and desmin-positive cells. (E) With a prolonged bevacizumab treatment of 70 days, the pericytes (desmin-positive cells) are alone able to revascularize the tumors. (F) In group ,F a second round of treatment does not prevent revascularization by CD31-positive cells. Scale bar (applies to all images): 100 μm.

Figure 4

Discontinuation of bevacizumab induces activation of alternative proangiogenic pathways. In order to understand the mechanisms of evasive resistance, including revascularization as a result of upregulation of alternative proangiogenic signals, we analyzed molecular changes of VEGF, PlGF, HGF, and HIF-1α in our three regimens. (A and B) Intratumor assay by ELISA. The concentration is expressed in pg/mg of proteins (pg/mg protein). (A) VEGF. (B) PlGF. (C, D and E) Plasma assay by radioimmunology. The concentration is expressed in ng/ml. (C) VEGF. (D) PlGF. (E) HGF. (F) HIF-1α; also intratumor assay by ELISA expressed in pg/mg protein. Data are presented as mean±SEM. .01<*P<.05; .001<**P<.01.

Figure 5

Discontinuous bevacizumab treatments do not modify EPC recruitment by SDF-1α but induces enrichment of CSCs. (A) The quantification by flow cytometry shows no differences between the groups in EPCs recruitments. (B) The SDF-1α assay also showed no difference between these groups. The assay concentration is expressed in pg/ml. We quantified, by flow cytometry, two types of CSC subpopulations, with ALDH1⁺, CD44⁺, CD166⁺ and ALDH1⁺, CD44⁺, CD166⁺, CD133⁺cell markers, respectively. (C) In the first regimen, there was no difference between the different groups, but when bevacizumab was stopped (group D), there was a significant increase of CSCs, and the same result was observed after a drug-break period (group F) with a significant increase compared to the group E. (D) As for the first subpopulation, there was no difference between the different groups for the D30 regimen. After withdrawal of bevacizumab (group D), there was a significant increase of the CSCs as in the case of the drug-break period (group F). Data are presented as mean±SEM. .01<*P<.05; .001<**P<.01.