1. Historical background The fact that osteosarcoma is highly malignant and the chance of survival never exceeds 20% after destructive surgery such as amputation [1] has prompted many scholars to search for effective anti-osteosarcoma drugs in an attempt to improve the prognosis of osteosarcoma patients through chemotherapy. 1961 Evans [2] reported 17 patients with stage III osteosarcoma (Enneking osteosarcoma staging system) using mitomycin In 1963, Sullivan [3] reported the efficacy of levulinic acid nitrogen mustard in osteosarcoma; subsequently, there were sporadic reports on the treatment of osteosarcoma with alkylating agents such as cyclophosphamide (CY), but the overall situation is that the efficacy of alkylating agents in the treatment of osteosarcoma is unstable and lacks clinical use. Friedman and Carter [4] reviewed the literature and concluded that the efficiency of alkylating agents for osteosarcoma was around 15%. In 1972 Cortes et al [5] reported that Adriamycin (ADM) treated 13 cases of stage III osteosarcoma, four of which obtained a good response; in 1972 Jaffe et al [6] were subjected to Djerassi with high-dose methotrexate and tetrahydrofolate relief (High-Dose Me-thotrexate with Citrovorum Factor “Rescue”, MTX+CFR) regimen for the treatment of progressive leukemia and lung cancer, 10 cases of stage III osteosarcoma were treated with HDMTX+CFR and four of them obtained significant responses. Subsequently, in 1974 Rosen et al [7] reported the use of sequential therapy with HDMTX+CFR and ADM to treat 13 cases of stage III osteosarcoma, with significant results achieved in 7 cases. Based on the exact efficacy of HDMTX+CFR and ADM on osteosarcoma and the fact that more than 80% of patients will develop lung metastases after osteosarcoma amputation, Rosen et al [8] and Jaffe et al [9] successively used these drugs alone or in combination for routine adjuvant therapy after osteosarcoma surgery, which significantly improved the prognosis of osteosarcoma patients and wrote a new chapter in the treatment of osteosarcoma. 2, adjuvant chemotherapy Rosen et al [7] found in the practice of applying HDMTX+CFR in the treatment of stage III osteosarcoma that the edema of metastases was reduced, pain was relieved, and the abnormally elevated alkaline phosphatase (Serum Alkaline Phosphatase, SAP) also decreased to the normal range after using the drugs. However, it was noted in clinical observations that in some cases, the normalized SAP would rebound within 2-3 weeks after administration, and drug resistance could occur with HDMTX+CFR alone, lacking further evidence of efficacy. Based on this clinical phenomenon, Rosen et al. combined HDMTX+CFR and ADM for the treatment of osteosarcoma, giving MTX or ADM twice a month, respectively, and treated a total of 15 patients with stage III osteosarcoma successively, resulting in an extension of the mean survival of stage III osteosarcoma from 3 months in the control group to 15 months. Also, because metastasis and recurrence of osteosarcoma mostly occur 9 to 10 months after surgery and the total course of treatment takes about 1 year, the total amount of ADM will exceed 900 mg/m2 if calculated according to this regimen, which will produce irreversible damage to the heart, and thus 1 CY was inserted between MTX and ADM to reduce the amount of ADM by: (1) VCR 1.5 mg/m2, MTX 200 mg/ kg.(2) CY 40-60mg/kg.(3) ADM 45mg/m2. 3 alternating with 2 weeks interval for 1 year, which was the earliest chemotherapy regimen for osteosarcoma, RosenT4 regimen [1]. Subsequently, several adjuvant chemotherapy regimens for osteosarcoma were reported to be applied successively [10]. As clinical studies progressed, cisplatinum (Cisplatinum,CDP), pediposide (Etoposide,VP16), isocyclophosphamide (Ifosfamide,IFO) and bleomycin, cyclophosphamide and actinomycin-D ( Dactiomycin) BCD, etc. The effective rate of osteosarcoma is 26% to 80% for single application and more effective for combined application [11]. Therefore, a series of multi-drug combination regimens have been developed accordingly, such as Rosen et al. T7 [12], T10 [13], T12 [14], Jaffe et al [15] and Bacci et al [11] osteosarcoma chemotherapy regimens. The main rationale is to combine drugs with different mechanisms of action, different toxicities, and definite effects on osteosarcoma according to certain rules to improve the effectiveness of chemotherapy [11]. However, when developing and implementing chemotherapy regimens, it is important not only to pay attention to the merits of the selected drugs, but also to understand and pay attention to the concept of Dose Intensity. 100% Dose Intensity means that the patient receives an accurate dose of the intended drug treatment within the specified time when receiving a certain chemotherapy regimen, and any factor that causes dose reduction or dosing delay will affect the final chemotherapy effect [11]. Bramwell et al [16] randomized 98 cases of osteosarcoma into 2 groups, one group received ADM (25 mg/m2 for 3 d continuously) and CDP (100 mg/m2 given once) for 6 courses, while the other group received HDMTX for 8 d followed by ADM and CDP, the single doses of ADM and CDP in the 2 groups were equal, and the total duration of chemotherapy Uchida et al [17] found that the dose intensity of the drugs had a significant impact on the prognosis of osteosarcoma after a 5-year follow-up of 67 patients with osteosarcoma. dose intensity had a greater effect on the prognosis of osteosarcoma than the addition of new drugs. In conclusion, without reducing the dose of a single drug per unit of time, the combination of drugs with different self-limiting toxicity and mechanism of action is beneficial to overcome the heterogeneity of tumor cells, reduce the generation of drug resistance, and improve the effect of chemotherapy [11]. 3, neoadjuvant chemotherapy Jaffe et al [18] reported in 1977 that 13 cases of osteosarcoma (4 cases of stage IIB and 9 cases of stage III) were treated with HDMTX once a week, and one case of osteosarcoma of the upper humerus received 4 weeks of preoperative HDMTX followed by intra-arterial perfusion of ADM for 6 h. In combination with local radiotherapy, it was found that the tumor shrank significantly, and angiography showed a decrease in neovascularization and tumor staining The tumor disappeared. After resection of the tumor, an artificial joint graft was performed, and the postoperative specimen was compared with the pre-chemotherapy biopsy specimen with significant tumor cell necrosis, fibrous membrane formation around the tumor foci, and near normal function of the reconstructed shoulder joint.Rosen et al [12] took advantage of the interval when patients with osteosarcoma were waiting for a special prosthesis to be made, and changed the T4 protocol from pure postoperative chemotherapy to preoperative initiation, which achieved significant results and allowed some patients to have their limbs The concept of neoadjuvant chemotherapy was gradually developed [13]. Neoadjuvant chemotherapy is the preoperative application of chemotherapy, and the revision of postoperative chemotherapy regimen is guided by the degree of response of the primary tumor foci to chemotherapeutic drugs, with the following specific reasons and advantages: (1) biological studies of tumors show that the sensitivity of micro metastases to chemotherapy is higher than that of relatively large metastases, and preoperative chemotherapy can enable patients to avoid the delay of rapid tumor growth and time due to the lowering of body immunity by surgical transfusion, etc. delay, and play the role of killing metastases in the first time; (2) kill the primary tumor foci as much as possible to make them shrink, which is conducive to limb-preserving surgery; (3) adjust individual chemotherapy regimen in time according to the response of the primary foci during chemotherapy; (4) screen out high-risk cases to receive intensive treatment before the tumor may recur or metastasize; (5) judge the prognosis, with good preoperative chemotherapy effect and high tumor The chance of tumor-free survival is relatively high for those who continue to receive chemotherapy after surgery with high cell necrosis rate [13]. The earliest neoadjuvant chemotherapy regimen was applied to the treatment of osteosarcoma by Rosen et al [12] in 1979, which consisted of HDMTX, ADM and BCD (T7 regimen), and achieved a survival rate of 70%, and after a longer follow-up, the results showed that the primary focus responded well to preoperative chemotherapy, and the prognosis of those with tumor cell necrosis rate greater than 90% was much better than those with less than 90%, and their The survival rates were 91% and 38%, respectively. Similarly, studies by Bramwell et al [16] and Provisor et al [19] have confirmed the correlation between the degree of preoperative tumor response to chemotherapy and prognosis. Adjusting the postoperative chemotherapy regimen according to the response of the tumor primary site to chemotherapeutic agents is one of the studies of interest, and Rosen et al [13] were the first to attempt this again by developing the T10 regimen in 1982. Preoperative chemotherapy was administered according to the T7 regimen, and postoperative chemotherapy was continued with the T7 regimen for those with tumor cell necrosis rates greater than 90%, and HDMTX was replaced by CDP in the regimen for those with tumor cell necrosis rates less than 90%, with a mean follow-up of 45 months and tumor-free survival rates of 75% and 76%, respectively, with no significant difference between the two. In the subsequent T12 regimen [14], the more toxic ADM and CDP in T10 were replaced by BCD, and if preoperative chemotherapy was not effective, ADM and CDP were applied for a longer period after surgery, and after 5 years of follow-up, the results showed that the overall efficacy of T10 and T12 was the same, and there was no difference between the results of those with good preoperative response and those with poor response. However, Meyers et al [20] and Provisor et al [19] failed to find that adjusting the postoperative chemotherapy regimen significantly improved the survival rate of those who were not sensitive to preoperative chemotherapy. Bacci et al. did not achieve similar results to Rosen et al. until 1991 and 1993 when new drugs such as VP16 and IFO were added to postoperative chemotherapy. 4. Preoperative route of administration Preoperative intra-arterial administration of tumor trophoblastic artery can make the primary focus obtain 1.5-4 times higher drug concentration than intravenous administration, enhance the local chemotherapy effect and facilitate the limb preservation, while the systemic blood concentration and intravenous administration of the same does not affect the accompanying systemic chemotherapy effect [21]. Jaffe et al [15] reported in 1985 a randomized comparison of the efficacy of arterial administration of MTX and CDP, and found that the CDP group responded well, with a tumor cell necrosis rate greater than 90% of 27%, while the CDP group reached 60%. Picci et al [22] conducted a comparative study of 79 cases of osteosarcoma with arteriovenous chemotherapy, in which patients received CDP, HDMTX and ADM chemotherapy successively. Bacci et al [21] performed preoperative intravenous HDMTX and intra-arterial CDP infusion chemotherapy for 127 cases of osteosarcoma, including 2 courses of intra-arterial HDMTX and a single intra-arterial CDP infusion for 72 h. The results showed that the preoperative chemotherapy was effective in 78% of the patients, while the other group was only 56%. In those who had good results with preoperative chemotherapy, ADM was added postoperatively in addition to the preoperative chemotherapy drugs, while in those who were not sensitive to preoperative chemotherapy, ADM and BCD were added. 63 cases (49%) survived for more than 6 years and 56 cases were treated with limb-sparing surgery without an increased rate of local recurrence. 66 cases (52%) had a long-term survival rate of 67% for those with tumor cell necrosis rates greater than 90%, which was significantly higher than that of 36% for those with tumor necrosis rates less than 90%. Uchida et al [17] followed up 67 cases of osteosarcoma treated with neoadjuvant chemotherapy for more than 4 years and found that the survival rate of the group with a single intra-arterial administration of CDP as part of preoperative chemotherapy was significantly higher than that of the groups with only intravenous MTX and ADM, 69.5% and 40.6%, respectively. The above results suggest that intra-arterial administration can obtain a higher tumor cell necrosis rate while still retaining the correlation between the degree of tumor cell necrosis and the prognosis of osteosarcoma, and that CDP is the preferred agent suitable for intra-arterial administration. Hyperthermic Isolated Limb Perfusion (HILP) allows for higher local drug concentrations in the tumor and can be combined with high temperature to maximize the killing effect on the primary site with less systemic toxicities. Guchelaar et al [23] reported that the local CDP concentration during HILP was 10-20 times higher than the systemic plasma CDP concentration, and 5 times higher than that of simple arterial administration, and the higher concentration was maintained throughout the HILP process. The author et al [24] started to use HILP in 1991 to treat osteosarcoma of the limb and obtained a high rate of tumor cell necrosis, and found that the local concentration was about 5 times higher than that of systemic chemotherapy by monitoring the blood platinum concentration, and that the systemic blood platinum concentration was close to that of systemic normal CDP chemotherapy after revascularization by adjusting the amount of drug-containing discarded fluid at the end of perfusion, while taking into account the The effect of systemic chemotherapy can be balanced with the effect of systemic chemotherapy. However, the local chemotherapy conditions in HILP are much better than those in systemic chemotherapy, so whether a high necrosis rate means a high survival rate remains to be further observed [25]. 5. Current problems and prospects In summary, with reasonable and aggressive chemotherapy, the limbs of about 80% of patients with osteosarcoma can be preserved, and the cure rate has increased from less than 20% with surgery alone to 50%-80% at present. However, no matter what kind of aggressive treatment is taken, about 40% of patients always develop lung metastasis at the time of consultation or during the treatment process, which eventually leads to treatment failure. Before the current major breakthroughs in immunotherapy and various biological therapies, it is decided how to enhance the effect of chemotherapy and further improve the cure rate of osteosarcoma based on the fact that chemotherapy can cure most of the cases is a top priority. Active discovery of new drugs and increasing drug intensity is one aspect of improving the effectiveness of chemotherapy, but more importantly, how to improve the sensitivity of osteosarcoma cells to chemotherapy. The main reason for drug resistance in some cases and eventual treatment failure may be related to multidrug resistance (MDR) of tumor cells. The detailed mechanism of MDR occurrence is not well understood, but it has been confirmed that it is related to the overexpression of mdrl gene and its product P-170 protein [26], which is essentially an energy-dependent efflux pump powered by ATP that transports specific molecules, including chemotherapeutic drugs, from intracellular to extracellular [27]. Although the relationship between MDR and tumor drug resistance is still debated, high levels of mdrl gene and high expression of P-170 protein have been found to be indeed present in drug-resistant osteosarcoma cells. Fortunately, the efflux pump effect of P-170 protein on chemotherapeutic drugs is specific and can be competitively inhibited by other classes of non-cytotoxic drugs known as MDR modulators. With the successful reversal of the MDR phenotype in in vitro cellular assays, the clinical use of MDR modulators to improve the efficacy of chemotherapy is not far away [28, 29].