CD13 is a therapeutic target in human liver cancer stem cells

Abstract

Cancer stem cells (CSCs) are generally dormant or slowly cycling tumor cells that have the ability to reconstitute tumors. They are thought to be involved in tumor resistance to chemo/radiation therapy and tumor relapse and progression. However, neither their existence nor their identity within many cancers has been well defined. Here, we have demonstrated that CD13 is a marker for semiquiescent CSCs in human liver cancer cell lines and clinical samples and that targeting these cells might provide a way to treat this disease. CD13+ cells predominated in the G0 phase of the cell cycle and typically formed cellular clusters in cancer foci. Following treatment, these cells survived and were enriched along the fibrous capsule where liver cancers usually relapse. Mechanistically, CD13 reduced ROS-induced DNA damage after genotoxic chemo/radiation stress and protected cells from apoptosis. In mouse xenograft models, combination of a CD13 inhibitor and the genotoxic chemotherapeutic fluorouracil (5-FU) drastically reduced tumor volume compared with either agent alone. 5-FU inhibited CD90+ proliferating CSCs, some of which produce CD13+ semiquiescent CSCs, while CD13 inhibition suppressed the self-renewing and tumor-initiating ability of dormant CSCs. Therefore, combining a CD13 inhibitor with a ROS-inducing chemo/radiation therapy may improve the treatment of liver cancer.

Figure 1. CD13 is a candidate marker of the SP fraction.

(A) The strategy used to identify cell-surface markers closely related to the SP fraction. We determined CD13 and CD31 as candidate markers for identifying SP cells. (B) Both CD13 and CD31 expression in HuH7 cells were compared in SP and non-SP cells by semiquantitative RT-PCR. Data represent mean ± SD from independent experiments of fractions differentially sorted by flow cytometry. *P < 0.01; **P = 0.076 versus non-SP fractions. The cut-off lines were determined using isotype controls. (C) Expression of CD13, CD133, and CD90 in HuH7 (upper panels) and PLC/PRF/5 cells (lower panels). Horizontal and vertical axes denote expression intensity. (D) The SP fraction is recognized as a “beak” appearing beside the G₁ phase fraction. The relationship between CD13⁺ and CD13^– cells and the SP fraction was studied using multicolor flow cytometry.

Figure 2. CD13 is a candidate marker of dormant to slow-growing CSCs.

(A) Dormant cells can be identified using the DNA-binding dye Hoechst 33342 and RNA-binding dye PY. Dormant cells contain lower RNA levels than G₁ phase cells. Combination analysis of the cell cycle with cell-surface markers CD13, CD133, and CD90 was performed with reserpine. The cut-off lines were determined using isotype controls. (B) Time-lapse cell-fate tracing of HuH7 cells. Cells were labeled with PKH26GL, isolated to their CD13⁺, CD13^–CD133⁺, and CD13^–CD133^– fractions, and traced for 238 hours. The dye-retaining cells can be identified as red-labeled cells (white arrow). Original magnification, ×20. (C) Proliferation assay of the CD13⁺CD133⁺, CD13^–CD133⁺, and CD13^–CD133^– fractions. Data represent mean ± SD from independent experiments of fractions differentially sorted by flow cytometry. *P < 0.05. (D) BrdU-retaining cells in serially transplanted control tumor specimens of HuH7 (6 weeks after BrdU injection) and PLC/PRF/5 (10 weeks after BrdU injection). The sections were stained with anti-CD13 (red), BrdU (green), and DAPI (blue). Top panels show lower magnification of the sections of HuH7 and PLC/PRF/5 (×10, HuH7 and left panel of PLC; ×20, right panel of PLC). The lower panels show high magnification (×40) of the place indicated by white arrows in the top panels.

Figure 3. CD13⁺ cells exist as a core in HCC spheres and produce CD90⁺ cells.

(A) Spheres established from HuH7 and PLC/PRF/5 cells were dissociated to single cells and the marker expressions were compared with control cells. Scale bars: 200 μm. (B) Expression analysis of primary human HCC cells (control) and spheres established from original human HCC cells (sphere). Scale bar: 200 μm. (C) The time-course expression analyses of sorted CD13⁺CD90^– cells (upper panels) and CD13^–CD90⁺ cells (lower panels) from PLC/PRF/5. The cut-off lines were determined using isotype controls.

Figure 4. CD13⁺ cells resist chemotherapy, and inhibition of CD13 elicits cellular apoptosis.

(A) The HuH7 and PLC/PRF/5 cells were treated with 0.1 μg/ml of DXR or 1 μg/ml of 5-FU for 72 hours. The changes in cell-surface markers were compared with controls. The percentages of CD13⁺CD133⁺ in HuH7 and CD13⁺CD90^– populations in PLC/PRF/5 are shown in figure. (B) Effect of CD13 inhibition on cell proliferation. HuH7 cells were treated with various concentrations of anti-human mouse IgG₁ CD13-neutralizing antibody. As a negative control, 10 μg/ml of anti-human mouse IgG₁ antibody was used. (C) Inhibition of CD13 induces cell apoptosis. Cells were treated with 1–20 μg/ml of CD13-neutralizing antibody or 50–500 μg/ml of ubenimex for 24 hours. Data show each case of 5 μg/ml of CD13-neutralizing antibody and 100 μg/ml of ubenimex treatment. (D) The effect of CD13-neutralizing antibody on DXR-R HuH7. The DXR-R clone was established with continuous treatment in 1 μg/ml of DXR and a selection of viable colonies. In 0.5 μg/ml of DXR, most control HuH7 cells die after 72 hours, whereas over 90% of DXR-R cells survive. The DXR-R HuH7 cells were cultured with 1–20 μg/ml of CD13-neutralizing antibody for 72 hours. Control, treated with 10 μg/ml of anti-human mouse IgG₁ antibody.

Figure 5. CD13 expression in clinical HCC samples with or without TAE.

(A) Expression analysis of clinical HCC samples. The nonhematopoietic Lin/CD45^– fraction was analyzed. The data show 2 typical TAE and non-TAE cases, nonhepatitis virus infection (NBNC; first row), nonhepatitis B but hepatitis C infection (NBC; second row), both hepatitis B and C infection (BC; third row), and another nonhepatitis B but hepatitis C infection (fourth row). The cut-off lines were determined using isotype controls. (B) Immunohistochemical analysis of HCC and normal liver samples stained with anti-human CD13 (red) and DAPI (blue) for the nucleus. Each middle panel shows a high magnification (×40) of the white dotted square in each left-hand column (×10). The right columns exhibit H&E stains (×10) of each adjacent frozen section. 3 typical samples from 6 HCC samples are represented. The white arrow indicates the fibrous capsule (Fc) in HCC samples, and tumor cells exist inside Fc. The upper 2 samples show HCC after TAE, and the middle samples are from no-TAE cases. In HCC samples, CD13 is expressed on the cell surface. The lower panels show immunohistochemical stains of normal liver obtained from surgical sections of colon cancer metastasis. Expression of CD13 in hepatic lobules is linear along the sinusoid. In the bile ducts, CD13 is expressed in an intraductal manner (lower left; bile duct, ×40). In some small canalicula present near the bile duct, CD13 is expressed on the cell surface (lower right; Glisson, ×40).

Figure 6. CD13 inhibition elicits cancer regression in vivo.

(A) HuH7-xenografted mice were treated with 5-FU or ubenimex for 3 days. The sections were stained with anti-human CD13 (red), Ki-67 (green), and DAPI (blue). Each right-hand panel shows a high magnification (×20, control and 5-FU; ×40, Ube) of the white dot square on the left (×10, control and 5-FU; ×20, Ube). White arrows: cellular clusters express CD13 but not Ki-67 (upper panels), residual Ki-67⁺ cancer cells (middle panels), and a residual CD13⁺ cell (lower panels). (B) PLC/PRF/5-xenografted mice were treated with 5-FU, ubenimex, and ubenimex plus 5-FU for 14 days. The black arrows indicate a small amount of residual cancer. The sections were stained with H&E (×10), anti-human CD13 (red), anti-human CD90 (green), and DAPI (blue) (×20, control, 5-FU, and Ube; ×40, Ube + 5-FU). Nonspecific and fragmented expression of CD13 (white arrow). In situ hybridization for DNA fragmentation (low and high magnification). Black dot-like structures indicate labeled DNA. (C) Tumors of control and ubenimex–plus–5-FU–treated mice. Black arrowheads indicate the tumor margin. (D) The relative tumor volumes (after treatment [mm³]/before treatment [mm³] × 100%) of the control, 5-FU, ubenimex, and ubenimex–plus–5-FU–treated mice. Data represent mean ± SD from independent experiments. *NS; **P < 0.01. (E) The CD13⁺ cell–enriched fractions obtained from 5-FU–treated mice were serially transplanted into secondary NOD/SCID mice. The mice were treated with ubenimex (Ube; n = 6) or received no treatment (control; n = 10) from the day after transplantation for 7 days. Tumor growth was observed for 3 weeks.

Figure 7. CD13⁺ cells contain lower levels of ROS than CD13^– cells.

(A) The expression of prooxidant DCF-DA in CD13⁺CD133⁺ and CD13^–CD133⁺ HuH7 cells and CD13⁺CD90^– and CD13^–CD90⁺ PLC/PRF/5 cells. Controls, treated with 10 μg/ml of mouse anti-human IgG. As positive controls, cells were treated with 100 μM of oxidant H₂O₂ for 2 hours. Cells were treated with 5 μg/ml of CD13-neutralizing antibody and 25 μg/ml of ubenimex for 4 hours. (B) The expression of ROS in the CD13⁺CD90^–, CD13^–CD90⁺, and CD13⁺CD90⁺ fractions of 2 clinical HCC samples. (C) The expression of the ROS scavenger pathway gene GCLM in isolated CD13⁺CD90^–, CD13⁺CD90⁺, CD13^–CD90⁺, and CD13^–CD90^– cells from PLC/PRF/5 and clinical HCC samples estimated by semiquantitative RT-PCR. (D) The time-course change of ROS expression in DXR or 5-FU treatment. Cells were treated with 1 μg/ml of DXR and 1 μg/ml of 5-FU continuously. After 3 hours and 48 hours of treatment, ROS levels in each population were measured.

Figure 8. High levels of ROS scavenger expression parallel DNA damage in CD13⁺ HCC cells.

(A) Isolated cell fractions of CD13⁺CD90^–, CD13⁺CD90⁺, CD13^–CD90⁺, and CD13^–CD90^– in PLC/PRF/5 and CD13⁺CD133⁺, CD13^–CD133⁺, and CD13^–CD133^– in HuH7 were irradiated with 4 Gy with or without antioxidant tempol. Data show the tail lengths in the alkaline comet assay of control (blue), 4 Gy irradiation (brown), and antioxidant tempol pretreated (green) cells. *P < 0.01. **NS. (B) HuH7 and PLC/PRF/5 cells were irradiated with 4 Gy, and time course change of gamma-H2AX expression in each population was assessed. Numbers indicate the percentage of gamma-H2AX in CD13⁺CD90^– PLC/PRF/5 and CD13⁺CD133⁺ HuH7 cells (red) and CD13^–CD90⁺ PLC/PRF/5 and CD13^–CD133⁺ HuH7 cells (blue) with ± SD. (C) HuH7 and PLC/PRF/5 cells were irradiated with 4 Gy, seeded in culture medium, and their expressions analyzed after 24 and 48 hours. Damaged and dead cells were eliminated with 7-AAD. The cut-off lines were determined using isotype controls.

Figure 9. The CD13⁺ CSCs of the liver generate genotoxic resistance through reduced levels of ROS (proposed schema).

(A) Results indicate that CD13⁺CD90^– CSCs of the liver are dormant and exhibit reduced intracellular ROS levels and, because of increased antioxidants, may result in resistance to genotoxic chemo/radiation therapy. On the other hand, CD13^–CD90⁺ CSCs actively proliferate and are sensitive to therapy. (B) Neutralization or inhibition of CD13 may result in an increase in intracellular ROS in CD13⁺CD90^– CSCs and induction of apoptosis.