Improved Therapeutic Effectiveness by Combining Recombinant CXC Chemokine Ligand 10 with Cisplatin in Solid Tumors

Cytotoxic chemotherapy has been the mainstay of medical approaches to the treatment of solid cancers. Cisplatin (DDP) remains the most widely used first-line element of cytotoxic chemotherapy to solid tumors (1). However, the efficacy of DDP based on the treatment is limited in curing most solid tumors due to dose-dependent toxicity. Therefore, several new therapeutic strategies under investigation involve modulation of cellular chemosensitivity, including the inhibition of tumor neovascularization, reversing tumor resistance, and increasing therapeutic effects of chemotherapy (2, 3). Angiogenesis inhibitors are likely to be relatively noncytotoxic, to exhibit a different spectrum of deleterious effects than those caused by the toxic agents (4, 5), and to work well in combination with chemotherapy (6–11).

To date, many agents including CXC chemokine have been identified as angiogenesis inhibitors. The CXC chemokines are a unique family of cytokines for their ability to behave in a disparate manner in the regulation of angiogenesis (12–17). A second structural domain within this family determines their angiogenic potential. Members that contain the three amino acids (Glu-Leu-Arg)/ELR immediately NH₂-terminal to the CXC motif (ELR⁺) are potent promoters of angiogenesis and stimulate the endothelial cell chemotaxis (e.g., interleukin-8). In contrast, the members that are induced by IFN and lack the ELR motif (ELR⁻) are potent inhibitors of angiogenesis, such as platelet factor-4 (PF4), monokine induced by IFN-γ (MIG, CXCL9), IFN-inducible T cell α chemoattractant (I-TAC, CXCL11), and CXC chemokine ligand 10 (CXCL10, IP-10).

CXCL10 is a potent inhibitor of angiogenesis and displays some thymus-dependent antitumor effect (16, 18–21). Therapy with CXCL10 is effective in reducing the rate of tumor growth, whereas it fails to induce tumor complete regression (21–24), which suggests that further treatment may require supplemental drugs targeting directly the tumor cells. Moreover, the recent findings from other groups indicated that to successfully counteract tumor progression, drugs inhibiting new vasculature formation should be employed in combination with traditional antitumor strategies, such as chemotherapy or radiotherapy (6–11, 25).

It has been shown that some CXC chemokines supported the survival of normal hematopoietic cells but not cancer cells and acted as chemoprotectant (26). Sarris et al. had also speculated that CXCL10 might be exploited to protect hematopoietic progenitors from the cytotoxic effects of chemotherapy (27). DDP-based chemotherapy often accompanied dose-limiting toxicity including bone marrow suppression. One approach is to maintain stem cells in a quiescent state during the periods of chemotherapy so that the cells have an increased period during which the repair of lethal damage may proceed before replication. For instance, evidence had indicated that normal hematopoietic cells treated by PF4 are still viable but at a relatively quiescent state. Thus, they are less sensitive to damage caused by cytotoxic drugs (26). Thus, we wonder whether it would improve the therapeutic efficacy of the antitumor by combining DDP with CXCL10. The present article showed that cooperated administration of DDP with CXCL10 improved therapeutic effectiveness in tumor-bearing immunocompetent animals.

Preparation of recombinant CXCL10. The nucleotide sequence encoding the mature region of murine CXCL10 cDNA (Genbank accession no. m33266; ref. 15) was PCR amplified from pBLAST-mIP-10 (Invivogen) and cloned into an expression plasmid pET-32a (Novagen). Recombinant mCXCL10 protein was generated in Escherichia coli. It was renatured by dropwise addition to 20 mmol/L phosphate buffer (pH 8.5) containing 0.1 mmol/L oxidized glutathione and 1 mmol/L reduced glutathione at 4°C. Purified mCXCL10 was prepared using a nickel-chelating column (Chelating Sepharose Fast Flow, Amersham Pharmacia Biotech, Uppsala, Sweden) on the chromatography system (AKTA, Amersham Pharmacia Biotech). The protein was eluted from the column with a linear gradient of imidazole, which was then dialyzed against the buffer containing decreased imidazole concentration. Finally, it was dialyzed by PBS. Recombinant proteins were further purified by ion exchange and gel filtration chromatography. The fusion protein has an enterokinase cleavage site between thioredoxin protein and CXCL10. Thioredoxin protein was cleaved from the fusion protein by enterokinase (Novagen). Fractions containing pure CXCL10 were identified by Tricine-PAGE and Western blot analysis as described previously (28). Membrane blots were probed with rabbit anti-murine CXCL10 antibody from Peprotech (Rocky Hill, NJ). Goat anti-rabbit immunoglobulin G linked to alkaline phosphatase (Sigma, St. Louis, MO), 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt, and p-nitroblue tetrazolium chloride color substrate (Bio-Rad, Richmond, CA) was used for detection. Protein concentration was determined by the Bradford assay using the Bio-Rad Protein Assay reagent (Bio-Rad) with bovine serum albumin (Sigma) as a standard. All protein preparation used in animal studies were filter sterilized, also tested for endotoxin, separated into aliquots, and frozen at −20°C.

Chemotaxis assay in vitro. Biological activity was evaluated by chemotaxis as previously described (29) with slight modification. T lymphocytes separated from mouse spleen stimulated for 2 days with 1 μg/mL phytohemagglutinin (Murex Diagnosis, Dartford, United Kingdom) and 400 units/mL interleukin-2 (Sigma) in RPMI 1640. Chemokine dilution medium supplemented with 1% low endotoxin bovine serum albumin was added to the bottom well of a 96-well Boyden microchambers chemotaxis plate (Neuroprobe). Fifty microliters of lymphocyte cells at the concentration of 5 × 10⁶ cells/mL were added on top of the membrane (5 μm pore size, polycarbonate filters, polyvinylpyrrolidone-free polycarbonate membranes). The chemotaxis plate was incubated at 37°C in an atmosphere containing 5% CO₂ for 4 hours and transferred to 4°C for 10 minutes before removing the membrane, fixed in 70% methanol, and stained for 5 minutes in 1% Coomassie brilliant blue. The number of cells that migrated to the lower surface was microscopically counted at six randomly chosen high power fields. Commercially available murine IFN-γ inducible protein 10 (IP-10) from Peprotech with biological activity was used as standard control. For the blocking experiments, bottom chamber was added with 30 μg/mL of rabbit anti-mCXCL10 antibody from Peprotech or control rabbit immunoglobulin G (Sigma). Three replicates were done for each treatment.

Tumor model. All studies involving mice were approved by the institute's Animal Care and Use Committee. CT26 colon adenocarcinoma and LL/2 Lewis lung cancer models were established in immunocompetent BALB/c and C57BL/6 mice at 6 to 8 weeks of age, respectively, as previously described (30).

Tumor growth inhibition study. Mice were weighed, coded, and divided into different groups when tumor was ∼90 mm³ after tumor cell inoculation. The experiment for the observation of tumor volume and survival advantage included 10 mice per group. Mice bearing established tumors were given systemically s.c. with mCXCL10 at 25 μg per kg per day, an effective dosage previously reported (20), once a day for 30 days in a volume of 0.1 mL at left flank, or appropriate controls injection in a volume of 0.1 mL PBS solution at the same time point. The regime of DDP (Qilu Pharmaceutical Co., Shandong, China) was cycled twice (5 mg/kg i.p. on days 14 and 21 after the initiation of CXCL10 administration) in 0.1 mL saline. Survival time and tumor volume were observed. Tumor size was determined by caliper measurement of the largest and perpendicular diameters every 4 days. Tumor volume was calculated according to the formula V = 0.52ab², where a is the largest superficial diameter and b is the smallest superficial diameter. The mice were sacrificed when they became moribund.

To evaluate the tumor weight and immunohistochemistry, the mice were killed by cervical dislocation on day 4 after the completion of treatment as described above. Excised tumors were weighed before their fixation in 10% formalin. In these models, the [mean excised treated-tumor weight divided by the mean excised control-tumor weight] × 100% was subtracted from 100% to give the tumor growth inhibition value for each group.

Alginate-encapsulated tumor cells assay. An alginate-encapsulated assay was done in vivo as previously described (31, 32). CT26 colon adenocarcinoma cells were resuspended in a 1.5% solution of sodium alginate (Sigma) and were added dropwise into a solution of 250 mmol/L calcium chloride. Alginate beads containing 1 × 10⁵ tumor cells per bead were formed. Four beads were then implanted s.c. into an incision made on the dorsal side of the BALB/c mice. Mice were subdivided into four groups and treated with PBS, CXCL10 alone, DDP alone, or CXCL10 + DDP (five mice per group), respectively. Treatment was initiated on day 2 with CXCL10 s.c. at 25 μg per kg per day continually for 14 days and/or DDP 5 mg/kg i.p. on days 4 and 10 after alginate beads grafting. Additional control animals were injected with PBS as described above. On day 15 after implanting, the mice were injected i.v. with 0.1 mL of a 100 mg/kg FITC-dextran (Sigma) solution through the tail vein. Alginate beads were rapidly removed and photographed 20 minutes after FITC-dextran introduction. The uptake of FITC-dextran was measured against a standard curve of FITC-dextran.

Histologic analysis. Sections of paraffin embedded from each group were stained with H&E. H&E section along the largest diameter of the tumor was scanned in the low field using the computer aided image analysis system Quantimet 600 and Qwin software (Leica, Benshaim, Germany). The total tumor area and necrotic tumor area were subsequently marked by the examiners on the screen. The areas were evaluated by the image analysis system, and the ratio tumor necrosis area to total tumor area was given in percentages. Leukocytes were quantitated as previously described (21). The organs such as lungs, liver, kidneys, etc. were also isolated for histology.

CXCL10-induced antiangiogenesis was determined by immunohistologic analysis of neovascularization in tumors. Tumors were embedded in paraffin, sectioned at 4 μm, deparaffinized in xylenes, and rehydrated through the graded ethanol into PBS. Antigen retrieval was done by microwaving in 10 mmol/L EDTA (pH 6.0); incubated with rat anti-mouse CD31 (platelet/endothelial cell adhesion molecule 1) polyclonal antibody (1:50; PharMingen, San Diego, CA), at 4°C, washed, incubated with biotinylated secondary antibody at room temperature for 30 minutes, washed, and incubated with avidin–biotin–horseradish peroxidase complex (Vectastain Elite; Vector Laboratories, Burlingame, CA), according to the manufacturer's instructions. Quantification was done as described by Vermeulen et al. (33). Microvessel counting was done at ×200. The results regarding angiogenesis were expressed as the absolute number of the microvessel per high-power field of three sections in each tumor.

Quantitative assessment of apoptosis. Tumor species were prepared as described above. Terminal deoxynucleotidyl transferase–mediated nick-end labeling staining was done using an in situ cell death detection kit (Roche Molecular Biochemicals) following the manufacturer's protocol. It is based on the enzymatic addition of digoxigenin nucleotide to the nicked DNA by terminal deoxynucleotidyl transferase. In tissue sections, four equal-sized fields were randomly chosen and analyzed. Density was evaluated in each field, yielding the density of apoptotic cells (apoptosis index).

Observation of potential toxicity. The toxicities of the treatment regimens were estimated by changes in animal body weight and the incidence of drug-associated death. Blood were obtained from the tail vein for complete blood count and enzyme analysis. Possible side effects of protein had been studied as previously described (30). The mice treated with recombinant CXCL10 s.c. without carcinomatosis have been investigated, in particular for potential toxicity, for >4 months. Gross measures, such as weight loss, ruffling of fur, life span, behavior, and feeding were studied. Tissues of heart, liver, spleen, lung, kidney, brain, bone marrow, etc. were also fixed in 10% neutral buffered formalin solution and embedded in paraffin. Sections of 4 μm were stained with H&E. Possible autoreactive antibody in serum of mice at various time points was determined by ELISA.

The combination of cytokines with chemotherapy may not be entirely innocuous because they may exacerbate an already present marrow reconstitutive defect such as residual cytopenias and marrow hypoplasia. Therefore, animals were subjected to complete peripheral blood counts and differentials. Evaluation of effects on hematopoiesis was conducted similar to previously described (34). Normal BALB/c and C57BL/6 mice (n = 5 per group) were given systemically s.c. with mCXCL10 at 10 or 25 μg per kg per day once daily for 30 days. The control group was injected with 0.1 mL PBS solution containing 1% bovine serum albumin. The mice were sacrificed on day 4 after terminating treatment. The tibiofibula were exposed by dissection and removed by cutting the bone as near to the malleolus and condyle as possible. The bone marrow was obtained by inserting a 23-gauge needle into the condyle end of the bone, and the cells were flushed out with 1 mL of PBS containing 1 unit/mL heparin. The leukocytes, erythrocytes, and platelets were enumerated. Cell cycle distribution was assessed as described by Vindelov et al. (35). Cells were processed and stained with propidium iodide, analyzed by flow cytometry (ESP Elite, Coulter). Cell cycle analysis was done using the sum of broadened rectangles model with the DNA software from Becton Dickinson (Mountain View, CA).

Statistical analysis. Data was assayed by ANOVA and Student's t test. For the survival time of animals, Kaplan-Meier curves were established for each group, and the survivals were compared by means of the log-rank test. Differences between means or ranks as appropriate were considered significant when yielding a P < 0.05.

Determination of biologically active CXCL10. Murine CXCL10 was expressed in E. coli as a fusion protein. Nickel chelataffinity chromatography was employed to purify large quantities of mCXCL10. Enterokinase was used to cleave the CXCL10 fusion protein. CXCL10 after purification and cleaviation has a predicted molecular mass of ∼9.5 kDa. Purity was >95% as determined by Tricine-PAGE (Fig. 1A). It was further verified by Western blot (Fig. 1B). Physiologic properties mimicking native CXCL10 was determined by its chemoattraction ability. Commercially available recombinant murine IP-10 from Peprotech was used as standard control. Recombinant CXCL10 in our experiment has a similar capacity of chemotaxis compared with purchased IP-10 in vitro. Chemotactic index was declined when anti-CXCL10 was given. Chemotaxis assay indicated that refolding of CXCL10 resulted in product with complete biological activity for further animal experiment (Fig. 1C).

Tumor growth inhibition. Tumor volume and life span of mice assay showed that both CXCL10 and DDP individually resulted in effective suppression of tumor growth. Combined treatment had a superior antitumor effect, resulting in 12- and 15-day (DDP 3.0 and 4.5 days) delay of tumor growth to reach a volume of 800 mm³ compared with PBS control in CT26 and LL/2 (Fig. 2A). In the two tumor models, control animals that received PBS treatment survived 32.1 and 32.7 days on average, respectively. In contrast, the combination of CXCL10 therapy with DDP resulted in a significant 3-fold increase in life span (P < 0.01, by log-rank test; Fig. 2B). Systemic therapy with CXCL10 + DDP resulted in apparent tumor inhibition versus PBS controls (P < 0.01) and DDP or CXCL10 alone (P < 0.05). The tumor growth inhibition was calculated using the mean excised tumor weight after completion of treatment for each group. Additionally, the appearance of lung metastases was significantly delayed by treatment with CXCL10 or CXCL10 + DDP in comparison with PBS control.

Alginate encapsulation assay. The inhibition of angiogenesis was confirmed in the alginate-encapsulated tumor cell assay. Figure 3A-D shows representative images of tumor vasculature in CT26 colon adenocarcinoma–encapsuled alginate. The vascular plexus of the tumor in PBS group formed a ladder-like pattern, which was richly morphologic and irregular (Fig. 3A). The treated tumor exhibited relatively little vascularity (Fig. 3B-D). Vascularization of beads over 15 days can then be measured by uptake of FITC-dextran into beads. Vascularization of alginate beads was apparently reduced, and FITC-dextran uptake was decreased 83% in CXCL10-treated mice compared with that in PBS controls (P < 0.05). No significant difference was observed between DDP group and PBS control mice (P = 0.17; Fig. 3E).

Immunolabeling of CD31. We further quantified vessel density as measures of angiogenesis by immunolabeling of CD31 in tissue sections. The most highly vascularized area of each tumor was identified on low power and five high-powered fields were counted in this area of greatest vessel density. The combination of CXCL10 and DDP apparently reduced the number of vessels compared with control groups, including PBS or DDP alone (P < 0.05). Angiogenesis was also inhibited in the treatment with CXCL10 alone compared with controls. No statistically significant differences were obtained between DDP and PBS group (P = 0.16 and 0.25 in LL/2 and CT26, respectively; Fig. 4).

Therapeutic effect on apoptosis. To explore the role of CXCL10 on apoptosis of tumor cells, tumors resected 4 days after the completion of treatment were subjected to terminal deoxynucleotidyl transferase–mediated nick-end labeling assays for the respective determination of apoptotic index. DDP or CXCL10 alone treatment affected the apoptosis rate of tumor cells, whereas the density of apoptotic cancer cells increased after the combined therapy (Fig. 5A). Data represent the mean apoptotic index ± SDs of cancer cells as percent normalized to apoptotic index of cancer cells (Fig. 5B).

Histologic analysis. Histologically, those CXCL10-treated tumors showed visible responses with homogeneous necrosis that was distinguishable in size and morphology from the several focal necroses of DDP-responsive tumors. Control tumors displayed little or no tumor tissue necrosis and had normal capillaries surrounding nests of confluent tumor cells. Analysis of the extent of tumor necrosis revealed that the coadministration of the two agents was clearly more effective, eliciting a 2.5-fold increase in tumor necrosis relative to single-element treatment (Fig. 6A-D). Leukocyte infiltration was next evaluated by histology within the tumor tissue. Leukocyte infiltration exhibiting clustering-like aggregation was apparently found in the margin of both CXCL10 alone and CXCL10 in conjunction with DDP group. Representative sections of PBS control and CXCL10 treated from CT26 neoplasm tissue were depicted (Fig. 6E-F). Lymphocyte infiltration was also remarkably elevated in central regions from CXCL10- or CXCL10 + DDP–treated groups. Representative sections of PBS control and CXCL10 treated from CT26 neoplasm tissue were depicted (Fig. 6G-H). Quantitation of tumor-infiltrating leukocytes revealed apparent difference in CXCL10 or CXCL10 + DDP relative to PBS treated (both P < 0.01).

Observation of potential toxicity. The systemic administration of cytokines can elicit antitumor effects, but the toxicities associated with this treatment occasionally resembled a state of severe infection and therefore can be limited (36, 37). Thus, CXCL10-treated animals without tumor burden were particularly investigated for potential toxicity of CXCL10 for >4 months. None of pathologic changes in liver, lung, kidney, etc. were found by microscopic examination after administration of CXCL10. No adverse consequences were showed in gross measures, such as weight loss, ruffling of fur, life span, behavior, or feeding. No autoreactive antibody in serum can be determined by ELISA. For evaluation of the effects of CXCL10 on hematopoiesis, animals were subjected to complete peripheral blood counts and differentials. Analysis of peripheral blood revealed that no significant difference was observed between DDP-treated and the combined treatment group. Our data suggested that traditional chemotherapeutic DDP in combination with CXCL10 was concurrently practicable with less host toxicity. In addition, bone marrow cells were enumerated and analyzed by microscope and cell cycles were analyzed by flow cytometry. No decreased or compensating hematopoiesis was found. There was also no apparent difference in the percent of G₀-G₁ or S-phase cell distribution between PBS-treated and CXCL10-treated (10 μg per kg per day) and CXCL10-treated (25 μg per kg per day) group (both P > 0.05, n = 5) in CT26 tumor. Neither accumulation of hematopoietic stem cells in the G₀ phase nor reduction in the percentage of cells in the S phase was observed. Similar results were also found in LL/2 neoplasm model (data not shown).

The generation of new blood vessels, angiogenesis, is a complex multistep process, which is tightly regulated by many positive and negative factors (2). Angiogenesis is important for normal embryonic development and for the development of pathologic conditions, such as cancer, rheumatoid arthritis, retinopathies, etc. (3). Several lines of direct and indirect evidence indicate that the growth and persistence of solid tumors and their metastasis are angiogenesis dependent (4). As a strategy for cancer therapy, antiangiogenic therapy attempts to stop new vessels from forming around a tumor and break up the existing network of abnormal capillaries that feeds the cancerous mass (4). A number of new angiogenesis inhibitors are currently being explored in a series of clinical trials and unfolded an inspiring perspective (38). Antiangiogenic therapy, although effective in inhibiting tumor growth, has not been shown tumoricidal in most studies (39). This therapeutic limitation can be overcome by combining angiogenesis inhibitors with cytotoxic therapies (6–11). CXCL10 as a potent angiostatic protein alone has also been used in antineoplasm in the tumor model in mice (20–23). The present study was designed to determine whether cisplatin potentiates the antitumor activity of CXCL10. The data in the present study showed that the combined treatment with CXCL10 and DDP led to an enhanced antitumor growth and the increased induction of apoptosis compared with CXCL10 and DDP alone. To our knowledge, this is the first time that CXCL10 and chemotherapy have been tested together and found to have the enhanced inhibitory effects on colon and lung carcinoma model in mice.

Although the exact mechanism by which the combination of CXCL10 with DDP can enhance the antitumor activity remained to be determined, the enhanced antitumor efficacy in vivo may in part result from the increased induction of the apoptosis in the combined treatment. This suggestion is supported by the present findings. The more apparent apoptotic cells in the tumors treated with CXCL10 + DDP was found compared with the treatment with CXCL10 or DDP alone or no-treatment groups. DDP has been effective in inducing apoptosis in variety of tumor cell lines (40–42). Its apoptosis-inducing effects are known to be correlated with DNA damage by forming DNA-Pt adducts and DNA strand breaks (40, 41). Recently, evidence indicated that CXCL10 reduced microvessel density, leading to the observed increase in both tumor cell apoptosis and necrosis (43). Our findings that the induction of apoptosis and necrosis within tumor by the inhibition of angiogenesis with CXCL10 in our experiments are coincided with previous reports (22). Thus, we may speculate that the potent antiangiogenesis activity by CXCL10 may play an important role in retarding or preventing adequate nourishment of tumors during their regrowth after a chemotherapeutic insult, resulting in tumor growth stasis. The inhibition of angiogenesis by CXCL10, therefore, is complementary to antitumor treatments, as was seen in our experiments. The other agents, including soluble vascular endothelial growth factor receptor (44), angiostatin (45), endostatin (46, 47), etc., has been also reported to inhibit tumor angiogenesis, to induce apoptosis, and to suppress tumor growth. The molecular mechanisms underlying the inhibition of angiogenesis, which in turn result in the induction of apoptosis in tumor cells by these agents, have yet to be elucidated fully (48). Furthermore, the other findings have shown that the combination of an antiangiogenic agent such as TNP-470 (7), vascular endothelial growth factor receptor-2 antibody (49), squalamine (8, 50), etc., with chemotherapy can enhance tumor growth inhibition. Taken together, these findings mentioned above also support our suggestion that the enhanced antitumor efficacy in the present study may in part result from the increased induction of the apoptosis in the combined treatment. In addition, the penetration of anticancer agents to cells distant from the vascular system is required for efficacy of cancer chemotherapy against solid tumors (51, 52). Recently, evidences showed that IFN-γ could sensitize chemotherapy by promoting penetration of chemotherapeutic drugs into the tissue gap of the solid tumor (53). Therefore, we can not ignore the possibility that IP-10/CXCL10 improved the penetration of DDP into tumor tissues responsible for the enhanced antitumor activity and the increased induction of apoptosis. These proposed mechanisms are persuasive, but further experiments in vitro and in vivo will be necessary to resolve the relative contribution of the hypothesized mechanism.

It has been reported that the intratumor injection of CXCL10 proteins or CXCL10-transduced cells led to reduced tumor growth and the potent inhibition of angiogenesis in a mouse models of lung cancer or melanoma (22, 24). We also found that the intratumoral administration of CXCL10 have recruited apparent mononuclear cells and profoundly inhibited angiogenesis, compared with systemic administration of CXCL10 (data not shown). However, the rationale for administering CXCL10 systemically in the present study is based on our effort to imitate clinical practice of most recombinant protein given as systemical rather than intratumoral. In fact, it is difficult for a clinical doctor to administer recombinant CXCL10 intratumorally in case of human lung or colon carcinoma, unless the metastatic lesions are found near body surfaces. Thus, intratumoral administration of CXCL10 may have limited clinical application in the future. Our findings suggest that the administration of recombinant CXCL10 systemically is effective in the inhibition of tumor growth. One could also achieve better antitumor response by combining CXCL10 with other chemotherapies or radiation.

It had been speculated that IP-10/CXCL10 may afford hematologic chemoprotection (26, 27), in addition to its antitumor properties for the treatment of cancers. We determined cell cycles of hematopoietic in the marrow by flow cytometry. No statistically significant difference in percentages of G₀-G₁, S, and G₂-M phase distribution were found between CXCL10 at 10 and 25 μg per kg per day and PBS control, which indicated that CXCL10 was well tolerated at the present dose. Either accumulation of hematopoietic stem cells in the G₀ phase or reduction in the percentage of cells in the S phase was not observed. Therefore, unless protection was mediated by a transient growth suppression that had no effect on the number of colonies seen in present concurrent administration rather than sequential, cell cycle redistribution does not seem to explain myeloprotective effects of CXCL10 mediated through inhibition of primitive progenitors from entering cycling phase.

In summary, our data suggest that the combination of CXCL10, a well-tolerated angiogenesis inhibitor, with DDP can enhance the antitumor activity, and that the enhanced antitumor efficacy in vivo may in part result from the increased induction of the apoptosis in the combined treatment. The present findings may be of importance to the further exploration of the potential application of this combined approach in the treatment of lung and colon carcinoma.