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In the present study, a patient-derived orthotopic xenograft (PDOX) model of recurrent cisplatinum (CDDP)-resistant metastatic osteosarcoma was treated with Salmonella typhimurium A1-R (S. typhimurium A1-R), which decoys chemoresistant quiescent cancer cells to cycle, and recombinant methioninase (rMETase), which selectively traps cancer cells in late S/G 2 , and chemotherapy. The PDOX models were randomized into the following groups 14 days after implantation: G1, control without treatment; G2, CDDP (6 mg/kg, intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, rMETase (100 unit/mouse, i.p., daily, for 2 weeks). G4, S. typhimurium A1-R (5 × 10 7 CFU/100 μl, i.v., weekly, for 2 weeks); G5, S. typhimurium A1-R (5 × 10 7 CFU/100 μl, i.v., weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks); G6, S. typhimurium A1-R (5 × 10 7 CFU/100 μl, i.v., weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks) and CDDP (6 mg/kg, i.p. injection, weekly, for 2 weeks). On day 14 after initiation, all treatments except CDDP alone, significantly inhibited tumor growth compared to untreated control: (CDDP: p = 0.586; rMETase: p = 0.002; S. typhimurium A1-R: p = 0.002; S. typhimurium A1-R combined with rMETase: p = 0.0004; rMETase combined with both S. typhimurium A1-R and CDDP: p = 0.0001). The decoy, trap and kill combination of S. typhimurium A1-R, rMETase and CDDP was the most effective of all therapies and was able to eradicate the metastatic osteosarcoma PDOX.

Gemcitabine (GEM) is first-line therapy for pancreatic cancer but has limited efficacy in most cases. Nanoparticle-albumin bound (nab)-paclitaxel is becoming first-line therapy for pancreatic cancer, but also has limited efficacy for pancreatic cancer. Our goal was to improve the treatment outcome in patient-like models of pancreatic cancer. We previously established patient-derived orthotopic xenografts (PDOX) pancreatic cancers from two patients. The pancreatic tumor was implanted orthotopically in the pancreatic tail of nude mice to establish the PDOX models. Five weeks after implantation, 50 PDOX mouse models were randomized into five groups of 10 mice for each pancreatic cancer PDOX: untreated control; GEM (100 mg/kg, i.p., once a week for 2 weeks); GEM + nab-PTX (GEM: 100 mg/kg, i.p., once a week for 2 weeks, nab-PTX: 10 mg/kg, i.v., twice a week for 2 weeks); S. typhimurium A1-R (5 × 10 7 CFU/100 μl, i.v., once a week for 2 weeks); GEM + S. typhimurium A1-R (GEM: 100 mg/kg, i.p., once a week for 2 weeks, S. typhimurium A1-R; 5 × 10 7 CFU/100 μl, i.v., once a week for 2 weeks). GEM + nab-PTX was significantly more effective than GEM alone in one PDOX model (p = 0.0004), but there was no significant difference in the other PDOX model. The combination of GEM + S. typhimurium A1-R regressed both PDOX models. These results show S. typhimurium A1-R can overcome the ineffectiveness or partial effectiveness of GEM in patient-like models of pancreatic cancer and demonstrate clinical potential for this combination.

Doxorubicin (DOX) is often first-line treatment of undifferentiated/unclassified soft tissue sarcoma (USTS). However, the DOX response rate for USTS patients is low. Individualized precision-medicine technology that could identify DOX responders as well as non-responders would be of high value to cancer patients. In the present study, we established 5 patient-derived orthotopic xenograft (PDOX) nude mouse models from 5 USTS patients and evaluated the efficacy of DOX in each PDOX model. USTS's were grown orthotopically in the right thigh of nude mice to establish the PDOX models. Two weeks after implantation, the mouse models were randomized into two groups of 8 mice each: untreated control; and DOX (3 mg/kg, i.p., once a week for 2 weeks). DOX showed significant growth inhibition in only 2 USTS PDOX models out of 5 (p = 0.0054, p = 0.0055, respectively) on day 14 after initiation. DOX was ineffective in the other 3 PDOX models. However, even in the DOX-sensitive cases, DOX could not regress the PDOX tumors responding to treatment. The present study has important implications since this is the first in vivo study to compare the DOX sensitivity for USTS on multiple patient tumors. We showed that only two of five USTS were responsive to DOX, despite DOX being first line chemotherapy for USTS. The 3 resistant cases should not be treated with DOX clinically, in order to spare the patients' unnecessary toxicity. This PDOX model is useful for precise individualized drug sensitivity testing, especially for rare heterogeneous recalcitrant sarcomas such as USTS.

Pancreatic cancer is a recalcitrant disease. Gemcitabine (GEM) is the most widely-used first-line therapy for pancreatic cancer, but most patients eventually fail. Transformative therapy is necessary to significantly improve the outcome of pancreatic cancer patients. Tumors have an elevated requirement for methionine and are susceptible to methionine restriction. The present study used a patient-derived orthotopic xenograft (PDOX) nude mouse model of pancreatic cancer to determine the efficacy of recombinant methioninase (rMETase) to effect methionine restriction and thereby overcome GEM-resistance. A pancreatic cancer obtained from a patient was grown orthotopically in the pancreatic tail of nude mice to establish the PDOX model. Five weeks after implantation, 40 pancreatic cancer PDOX mouse models were randomized into four groups of 10 mice each: untreated control (n = 10); GEM (100 mg/kg, i.p., once a week for 5 weeks, n = 10); rMETase (100 units, i.p., 14 consecutive days, n = 10); GEM+rMETase (GEM: 100 mg/kg, i.p., once a week for 5 weeks, rMETase: 100 units, i.p., 14 consecutive days, n = 10). Although GEM partially inhibited PDOX tumor growth, combination therapy (GEM+rMETase) was significantly more effective than mono therapy (GEM: p = 0.0025, rMETase: p = 0.0010). The present study is the first demonstrating the efficacy of rMETase combination therapy in a pancreatic cancer PDOX model to overcome first-line therapy resistance in this recalcitrant disease.

Gastrointestinal stromal tumor (GIST) with a mutation in exons 11 and 17 of c-kit is a rare type of sarcoma. The aim of this study was to determine drug sensitivity for a regionally-recurrent case of GIST using a patient-derived orthotopic xenograft (PDOX) model. The PDOX model was established in the anterior wall of the stomach. GIST PDOX models were randomized into 5 groups of 6 mice each when the tumor volume reached 60 mm 3 : G1, control group; G2, imatinib group (oral administration (p.o.), daily, for 3 weeks); G3, sunitinib group (p.o., daily, for 3 weeks); G4, regorafenib (p.o., daily, for 3 weeks); G5, pazopanib (p.o., daily, for 3 weeks). All mice were sacrificed on day 22. Tumor volume was evaluated on day 0 and day 22 by laparotomy. Body weight were measured 2 times per week. Though regorafenib is third-line therapy for GIST, it was the most effective drug and regressed the tumor significantly (p < 0.001). Sunitinib suppressed tumor growth compared to the control group (p = 0.002). Imatinib, first-line therapy for GIST, and pazopanib did not have significant efficacy compared to the control group (p = 0.886, p = 0.766). The implications of this result is discussed for GIST patients.

The elevated methionine (MET) use by cancer cells is termed MET dependence and may be the only known general metabolic defect in cancer. Targeting MET by recombinant methioninase (rMETase) can arrest the growth of cancer cells in vitro and in vivo. We previously reported that rMETase, administrated by intra-peritoneal injection (ip-rMETase), could inhibit tumor growth in a patient-derived orthotopic xenograft (PDOX) model of a BRAF-V600E mutant melanoma. In the present study, we compared ip-rMETase and oral rMETase (o-rMETase) for efficacy on the melanoma PDOX. Melanoma PDOX nude mice were randomized into four groups of 5 mice each: untreated control; ip-rMETase (100 units, i.p., 14 consecutive days); o-rMETase (100 units, p.o., 14 consecutive days); o-rMETase+ip-rMETase (100 units, p.o.+100 units, i.p., 14 consecutive days). All treatments inhibited tumor growth on day 14 after treatment initiation, compared to untreated control (ip-rMETase, p<0.0001; o-rMETase, p<0.0001; o-rMETase+ip-rMETase, p<0.0001). o-rMETase was significantly more effective than ip-rMETase (p = 0.0086). o-rMETase+ip-rMETase was significantly more effective than either mono-therapy: ip-rMETase, p = 0.0005; or o-rMETase, p = 0.0367. The present study is the first demonstrating that o-rMETase is effective as an anticancer agent. The results of the present study indicate the potential of clinical development of o-rMETase as an agent for chronic cancer therapy and for cancer prevention and possibly for life extension since dietary MET reduction extends life span in many animal models.

A metastatic melanoma obtained from the right chest wall of a patient was previously established orthotopically in the right chest wall of nude mice as a patient-derived orthotopic xenograft (PDOX) model. We previously showed that the combination of tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) and chemotherapy was highly effective against the melanoma PDOX. In the present study, we investigated the mechanism of the high efficacy of this combination. Two weeks after implantation, 40 PDOX mouse models were randomized into 4 groups of 10 mice each: untreated control (n = 10); treated with S. typhimurium A1-R (5 × 10 7 CFU/100 μl, i.v., once a week for 2 weeks, n = 10); treated with temozolomide (TEM) (25 mg/kg, p.o. for 14 consecutive days) combined with S. typhimurium A1-R (5 × 10 7 CFU/100 μl, i.v., once a week for 2 weeks, n = 10); treated with vemurafenib (VEM) (30 mg/kg, p.o., for 14 consecutive days) combined with S. typhimurium A1-R (5 × 10 7 CFU/100 μl, i.v., once a week for 2 weeks) (n = 10). On day 14 from initiation, all treatments significantly inhibited tumor growth compared with untreated control (S. typhimurium A1-R: p < 0.01; TEM combined with S. typhimurium A1-R: p < 0.01; VEM combined with S. typhimurium A1-R: p < 0.01). Combination therapy with S. typhimurium A1-R was significantly more effective on tumor growth than S. typhimurium A1-R alone (with TEM: p < 0.01; with VEM: p < 0.01). Combination therapy significantly increased S. typhimurium A1-R tumor targeting alone (S. typhimurium A1-R + TEM: p < 0.01, S. typhimurium A1-R + VEM: p < 0.01), relative to S. typhimurium A1-R alone, respectively. In conclusion, chemotherapy drugs promoted targeting of S. typhimurium A1-R of melanoma, thereby enhancing efficacy against the melanoma PDOX.

Previously, a patient-derived orthotopic xenograft (PDOX) model was established with a lung metastasis from an osteosarcoma patient which developed after adjuvant cisplatinum (CDDP) treatment. In this model, we previously demonstrated the efficacy of trabectedin (TRAB) and temozolomide (TEM) compared with CDDP. In the present report, osteosarcoma tissue was implanted orthotopically in the distal femur of mice which were randomized into the following groups when tumor volume reached approximately 100 mm 3 ; On day 14 after initiation of treatment, all but CDDP significantly inhibited tumor volume growth compared with untreated controls. Control (G1): 793.7 ± 215.0 mm 3 ; CDDP (G2): 588.1 ± 176.9 mm 3 ; Salmonella typhimurium A1-R (S. typhimurium A1-R) intravenous (i.v.) (G3): 269.7 ± 72.7 mm 3 ; S. typhimurium A1-R intra-arterial (i.a.) (G4): 70.2 ± 18.9 mm 3 (CDDP: p = 0.056; S. typhimurium A1-R i.v.: p = 0.0001; S. typhimurium A1-R i.a.: p = 0.00003, all vs. untreated controls). i.a. administration of S. typhimurium A1-R was significantly more effective than either CDDP (p = 0.00007), or i.v. administration of S. typhimurium A1-R (p = 0.00007) and significantly regressed the tumor volume compared with day 0 (p = 0.001). The new model of i.a. administration of S. typhimurium A1-R has great promise for the treatment of recalcitrant osteosarcoma.

William B. Coley developed bacterial therapy of cancer more than 100 years ago and had clinical success. James Ewing, a very famous cancer pathologist for whom the Ewing sarcoma is named, was Coley's boss at Memorial Hospital in New York and terminated Coley's bacterial therapy of cancer. A tumor from a patient with soft-tissue Ewing's sarcoma, who failed doxorubicin (DOX) therapy, was previously implanted in nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. In the present study, the Ewing's sarcoma PDOX was treated with tumor-targeting S. typhimurium A1-R expressing green fluorescent (GFP), alone and in combination with DOX. S. typhimurium A1-R-GFP was detected in the tumors after intratumor (i.t.) or intravenous (i.v.) injection. The combination of S. typhimurium A1-R and DOX significantly reduced tumor weight (37.8 ± 15.6 mg) compared to the untreated control (73.8 ± 10.1 mg, P < 0.01). S. typhimurium A1-R monotherapy-treated tumors tended to be smaller (50.9 ± 17.8 mg, P = 0.051). DOX monotherapy did not show efficacy (66.3 ± 26.4 mg, P = 0.82), as was the case with the patient. The PDOX model faithfully replicated the DOX resistance the Ewing's sarcoma had in the patient. S. typhimurium A1-R converted the Ewing's sarcoma from DOX resistant to sensitive. One can only wonder how bacterial therapy and immunotherapy of cancer would have developed over the past 80 years if Ewing did not stop Coley.

Gastric leiomyosarcoma is a recalcitrant cancer and the chemotherapy strategy is controversial. The present study used a patient-derived orthotopic xenograft (PDOX) nude mouse model of gastric leiomyosarcoma to identify an effective therapeutic regimen to develop individualized precision medicine for this disease. The gastric leiomyosarcoma obtained from a patient was first grown in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP) to stably label the tumor stroma. The RFP-expressing tumor was then passaged orthotopically in the gastric wall of non-transgenic nude mice to establish an imageable PDOX (iPDOX) model. The bright fluorescent tumor was readily imaged over time to determine drug efficacy. Four weeks after implantation, 70 PDOX nude mice were divided into 7 groups: control without treatment (n = 10); doxorubicin (DOX) (2.4 mg/kg, intraperitoneally (i.p.), once a week for 2 weeks, n = 10); gemcitabine (GEM)/ docetaxel (DOC) (GEM: 100 mg/kg, DOC: 20 mg/kg, i.p., once a week for 2 weeks, n = 10); cyclophosphamide (CPA) (140 mg/kg, i.p., once a week for 2 weeks, n = 10); temozolomide (TEM) (25 mg/kg, orally, daily for 14 consecutive days, n = 10); yondelis (YON) (0.15 mg/kg, i.v., once a week for 2 weeks, n = 10); pazopanib (PAZ) (100 mg/kg, orally, daily for 14 consecutive days, n = 10). On day 14 from initiation of treatment, all treatments except PAZ significantly inhibited tumor growth compared with untreated control (DOX: p < 0.01, GEM/DOC: p < 0.01, CPA: p < 0.01, TEM: p < 0.01, YON: p < 0.01) on day 14 after initiation. In addition, only GEM/DOC was more significantly effective than DOX (p < 0.05). GEM/DOC could regress the leimyosarcoma in the PDOX model and has important clinical potential for precision individual treatment of leiomyosarcoma patients.

Rhabdomyosarcoma (RMS) is a rare mesenchymal tumor. The aim of the present study was to develop a patient-derived orthotopic xenograft (PDOX) mouse model of RMS and compare the PDOX model to a subcutaneous (s.c.)-transplant model. A patient RMS from a striated muscle was grown orthotopically in the right biceps femoris muscle and right quadriceps muscle of nude mice to establish a PDOX model, as well as under the skin to establish an s.c., Model: PDOX tumors grew at a statistically-significant faster rate compared to the s.c. tumors. Recurrence after surgical resection occurred only in PDOX tumors, not in the s.c., Model: Histologically, only the PDOX model was shown to be invasive. In conclusion, these results indicate that the PDOX model of adult RMS is malignant and the subcutaneous model is benign. These results emphasize that a proper tumor microenvironment is necessary for patient-like behavior of a tumor in a mouse model.