ANTICANCER RESEARCH 25: 947-954 (2005) Osteogenic Progenitor Cell Potency After High-Dose Chemotherapy (COSS-96) M. JÄGER, A. SCHULTHEIS, B. WESTHOFF and R. KRAUSPE Department of Orthopaedics, Heinrich-Heine University Hospital Duesseldorf, D-40225 Duesseldorf, Germany Abstract. Background: Since the first trial of chemotherapy in have demonstrated that a reduction in osteoblast numbers patients with osteosarcoma the survival rate has gradually results in diminished bone formation, too (1). As a improved. For more than two decades, most osteosarcoma complication of chemotherapy / corticosteroids for the patients from Germany, Austria and Switzerland have been treatment of acute lymphoblastic leukemia (ALL) and other treated according to the protocols of the Cooperative malignancies like osteosarcoma during childhood, avascular Osteosarcoma Study Group (COSS). The uniform treatment osteonecrosis appears in up to 30% of the patients (2, 3). concept of a high-dose polychemotherapy pre- and Moreover, chemotherapy may lead to a reduction of postoperatively improved the survival rate of these patients osteoprogenitor cells and affect osteoblast regeneration significantly. One severe side-effect of COSS chemotherapy is potency in bone marrow and bone, which was measured by multiple osteonecrosis. Patients and Methods: In this study the a reduction of colony forming units in vitro (4). osteogenic stem cell potency of three different tissue types was For more than two decades, most osteosarcoma patients elucidated after COSS-96 chemotherapy (high-risk arm). from Germany, Austria and Switzerland have been treated Mononuclear cells were obtained from the periosteum, cartilage on protocols of the Cooperative Osteosarcoma Study Group and bone marrow of a 17-year-old female with a chondroblastic (COSS). The uniform treatment concept of a high-dose pre- osteosarcoma. The cells were cultivated for 4 weeks in standard and postoperative polychemotherapy improved the survival medium and stimulated for osteogenic differentiation after the rate of these patients significantly (5, 6). The preoperative second passage with dexamethasone, glycerolphosphate and neoadjuvant chemotherapy protocol includes the application ascorbine acid. Two weeks later, the cell cultures were analysed of doxorubicin or adriamicin, methotrexate, cisplatin and with respect to cell morphology and immunochemical stainings. ifosfamid. Results: All cells cultures showed an osteoblastic regeneration Particularly the anthracycline doxorubicin is responsible potential measured by osteocalcin (OC), osteopontin (OP) and for bone marrow depressions during and after alkaline phosphatase (ALP) expression. Compared to other chemotherapy. Its toxic effect is based on an intercalation donor tissues and localizations, the fibula periosteum showed between the nucleotides and in inhibition of topoisomerase siginificantly higher osteoblast rates in vitro, whereas collagen II, which results in an inhibition of DNA and RNA II, CD34 and CD45 were not expressed in any culture. synthesis, especially during the S-phase of the cell cycle (7). Conclusion: The results of this study demonstrate the survival of It can be assumed that those cells which have a high mesenchymal progenitor cells in bone marrow during COSS-96 proliferation rate, such as tumor and/or stem cells, are more polychemotherapy, which allows for an osteogenic regeneration sensitive than other cells. in vitro and potentially in vivo. Besides other tissues, multipotent mesenchymal stem cells (MSC) are localized in human bone marrow, the Clinical studies suggest that combination chemotherapy cambium of periosteal tissue (8) and in small numbers, in adversely affects bone metabolism, while in vitro studies joint cartilage, too (9). To date it is not known to what extent a COSS-96 chemotherapy may affect these stem cell- containing tissues. In contrast to other investigators, who developed appropriate in vitro screening systems to evaluate Correspondence to: Marcus Jäger, M.D., Department of the clinical response and efficiency of chemotherapeutics by Orthopaedics, Heinrich-Heine University Duesseldorf, Moorenstr. 5, D-40225 Duesseldorf, Germany. Tel: +49 (0)211-81-7961, Fax: cell culture systems (10, 11, 12), we elucidated the in vitro +49 (0)211-81-6281, e-mail: [email protected] regeneration potential of mesenchymal stem cells after in vivo neoadjuvant chemotherapy based on the COSS-96 Key Words: Osteosarcoma, chemotherapy, stem cell, osteoblast. protocol. 0250-7005/2005 $2.00+.40 947 ANTICANCER RESEARCH 25: 947-954 (2005) Figure 1. Preoperative X-rays in two planes (a) and MRI scans (b) of the left lower leg showing signs of a malignant solid tumor in the proximal tibia corresponding to an osteosarcoma. Patients and Methods dexametasone, beta-glycerolphosphate and ascorbine acid (DAG) for another 2 weeks, as described previously by Pittenger et al. (13). Mononuclear cells were obtained from a 17-year-old female As osteoblast specific markers, alkaline phosphatase (ALP), volunteer donor with a chondroblastic osteosarcoma of the left osteocalcin (OC) and osteopontin (OP) were used, whereas CD105 proximal tibia 2 months after COSS-96 polychemotherapy (high-risk antigen (endoglin) served as a mesenchymal stem cell marker. A von arm, doxorubicin, methotrexate, cisplatin and ifosfamid) (Figures 1 Kossa staining showed in vitro calcification. Furthermore collagen II and 2). All experiments were carried out under written consent was measured to detect chondroblastic differentiation, while CD34 / according to the Declaration of Helsinki for good scientific practice. CD45 served to evaluate for hematopoietic cell differentiation. The No metastases were detected by bone scan or MRI scans, but specimens were fixed with 5% paraformaldehyde at 4ÆC for 30 min, there was infiltration of the neurovascular bundle. The patient rinsed in phosphate-buffered saline (PBS) and dehydrated in graded underwent knee amputation (exarticulation) with a wide tumor-free alcohols. Endogenous peroxidases of the specimens were blocked by resection line confirmed by the pathologist. Tumor-free cartilage and 3% perhydrol-isopropanol solution. After rinsing in Tris-buffer, the periosteoum strips of 0.5 x 0.5 mm in length were obtained from the cell culture dishes were incubated with primary antibodies against ankle joint (tibia, fibula and talus of the amputate). Furthermore, CD-antigens with further incubation at 4ÆC for 12 h. For optical spongious bone marrow was taken from the distal tibia, fibula and visualization, the second antibody system with avidin-biotin-complex talus. The tissue specimens were cultivated for primary explant and 3,3-diaminobenzidine was used. ALP activity was measured by cultures in DMEM (PAA, Cölbe, FRG) with 10% FCS (FCS, direct substrate incubation (SK-5.200, Vector, Burlingame CA, Biochrome, Berlin, FRG), at 37.0ÆC and 5.0% CO2 for 2 weeks. USA) for 30 min at room temperature (RT). The cell cultures were Afterwards the strips / solid tissue samples were removed and the analysed blind by an independent observer via episcopic light adherent cells were passaged (0.05% trypsin/EDTA). The cultures microscopy (Axiovert 200, Zeiss, FRG) in combination with a were expanded for two weeks until a confluent monolayer occurred. computer imaging picture analysis system (Axiovision, Zeiss), after After the second passage, osteogenic stimulation was initiated by an in vitro follow-up of 6 weeks in total. 948 Jäger et al: Osteoprogenitor Cells After Chemotherapy Figure 2. Chemotherapy protocol of COSS-96 scheme. A: Adriamycin or Doxorubicin (90 mg / m2 over 48 h i.v.), M: Methotrexate with Folin acid- Rescue (12 g / m2 within 4 h i. v., Folin-Rescue 24 h after Methotrexate application), I: Ifosfamid (3 g / m2 over 1 h i. v.), P: Cisplatin (120 mg / m2 over 72 h i. v.), C: Carboplatin (150 mg / mÇ over 1 h i. v.), E: Etoposid (150 mg / m2 over 1 h i. v.). Low risk: volume of tumor ≤ 150 ml in regression IÆ or IIÆ. High risk: volume of tumor > 150 ml in regression VÆ or VIÆ. Standard risk: patients with a volume of tumor ≤ 150 ml in regression IIIÆ-VIÆ or > 150 ml in regression IÆ-IVÆ. For statistical analysis the Student’s t-test for independent Compared to other cell cultures, the cells isolated from statistical groups was used. P<0.01 was rated highly statistical the periosteum of the fibula showed the highest number of significant and p<0.05 statistically significant, whereas p>0.05 OC- and OP-positive cells, which characterize the mature showed no significance. The average values (X) and standard osteoblast. Corresponding to the potential of osteoblast deviations (SD) served as descriptive parameters. differentiation, CD105+ cells were present in all cell Results cultures. Calcification was found in all OP- and OC-positive cultures, demonstrated by von Kossa staining. Furthermore, all cultures showed no collagen II expression in The results of this study show clearly that, after COSS-96 immunocytochemistry after osteoblastic stimulation with chemotherapy, an osteogenic stem cell differentiation is DAG. Figure 3 illustrates the immunocytochemical staining maintained. By primary cell cultures derived from cartilage, behaviour for osteoblasts, chondroblasts, hematopoietic and periosteum and bone marrow in different organs, we could mesenchymal progenitor cells in vitro, whereas Figures 4 demonstrate a high osteoblast differentiation rate (Figure and 5 show the quantification of the cellular differentiation. 3). There were significant