Synergy of oral recombinant methioninase (rMETase) and 5-fluorouracil on poorly differentiated gastric cancer.

Gastric cancer is highly malignant and recalcitrant to first line chemotherapies that include 5-fluorouracil (5-FU). Cancer cells are addicted to methionine for their proliferation and survival. Methionine addiction of cancer is known as the Hoffman effect. Methionine restriction with recombinant methioninase (rMETase) has been shown to selectively starve cancer cells and has shown synergy with cytotoxic chemotherapy including 5-FU. The present study aimed to investigate the efficacy of rMETase alone and the combination with 5-FU on poorly differentiated human gastric cancer cell lines (MKN45, NUGC3, and NUGC4) in vitro and vivo. rMETase suppressed the tumor growth of 3 kinds of poorly differentiated gastric cancer cells in vitro. The fluorescence ubiquitination-based cell cycle indicator (FUCCI) demonstrated cancer cells treated with rMETase were selectively trapped in the S/G2 phase of the cell cycle. In the present study, subcutaneous MKN45 gastric cancer models were randomized into four groups when the tumor volume reached 100 mm3: G1: untreated control; G2: 5-FU (i.p., 50 mg/kg, weekly, three weeks); G3: oral-rMETase (o-rMETase) (p.o., 100 units/body, daily, three weeks); G4: 5-FU with o-rMETase (5-FU; i.p., 50 mg/kg, weekly, three weeks o-rMETase; p.o., 100 units/body, daily, three weeks). All mice were sacrificed on day 22. Body weight and estimated tumor volume were measured twice a week. 5-FU and o-rMETase suppressed tumor growth as monotherapies on day 18 (p = 0.044 and p = 0.044). However, 5-FU combined with o-rMETase was significantly superior to each monotherapy (p < 0.001 and p < 0.001, respectively) and induced extensive necrosis compared to other groups. The combination of 5-FU and o-rMETase shows promise for transformative therapy for poorly differentiated gastric cancer in the clinic.

Imaging the interaction of αv integrin-GFP in osteosarcoma cells with RFP-expressing host stromal cells and tumor-scaffold collagen in the primary and metastatic tumor microenvironment.

Human osteosarcoma 143B cells were previously stably transfected with an αv integrin green flourescent protein (GFP) vector. 143B cells expressing αv integrin-GFP were transplanted orthotopically in the tibia of transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary tumors acquired RFP-expressing stroma and were passaged orthotopically in the tibia in noncolored nude mice, which maintained the RFP stroma. The interaction of αv integrin-GFP expression in 143B cells with RFP-expressing host stromal cells was observed by confocal microscopy using the Olympus FV1000. Collagen fibers were imaged simultaneously in reflectance mode. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) which persisted even 3 weeks after passage to nontransgenic nude mice. CAFs expressing RFP were aligned between collagen fibers and cancer cells expressing αv integrin-GFP. Six weeks after transplantation, pulmonary metastases expressing αv integrin-GFP could be identified. TAMs expressing RFP accompanied metastasized osteosarcoma cells expressing αv integrin-GFP in the lung. The current study demonstrates the importance of αv integrin interaction with stromal elements in osteosarcoma.

Gemcitabine combined with docetaxel precisely regressed a recurrent leiomyosarcoma peritoneal metastasis in a patient-derived orthotopic xenograft (PDOX) model.

There are no effective treatments for leiomyosarcoma (LMS) spreading intraabdominally. The aim of this study was to develop precision chemotherapy for recurrent peritoneal LMS metastases in a patient-derived orthotopic xenograft (PDOX) model. The LMS PDOX models were established orthotopically on the dome of the bladder of nude mice. The LMS PDOX models were randomized into 6 groups when the tumor volume reached 80 mm3: G1: untreated control; G2: doxorubicin (DOX) (DOX: i.p., 3 mg/kg, weekly, 3 weeks); G3: DOX combined with olaratumab (OLA) (DOX: i.p., 3 mg/kg, weekly, 3 weeks; OLA: i.p., 40 mg/kg, 3 times/week, 3 weeks); G4: gemcitabine (GEM) combined with docetaxel (DOC) (GEM: i.p., 100  mg/kg, weekly, 3 weeks; DOC: i.p., 20  mg/kg, weekly, 3 weeks); G5: pazopanib (PAZ) (PAZ: p.o., 100  mg/kg, daily, 3 weeks); G6: palbociclib (PAL) (PAL: p.o., 100  mg/kg, daily, 3 weeks). All mice were sacrificed on day 22. Body weight was assessed twice a week. Tumor volume was measured on day 0 and day 22. Although all regimens had a significant efficacy compared to the untreated group (P < 0.001), only GEM combined with DOC regressed the tumor significantly (P < 0.001), suggesting GEM combined with DOC has clinical potential for this LMS patient.

A patient-derived orthotopic xenograft (PDOX) nude-mouse model precisely identifies effective and ineffective therapies for recurrent leiomyosarcoma.

Leiomyosarcoma is a rare and recalcitrant disease. Doxorubicin (DOX) is usually considered first-line treatment for this disease, but frequently is ineffective. In order to individualize therapy for this and other cancers, we have developed the patient-derived orthotopic xenograft (PDOX) mouse model. In the present study, we implanted a recurrent leiomyosarcoma from a resected tumor from the patient's thigh into the femoral muscle of nude mice. The following drugs were tested on the leiomyosarcoma PDOX model: DOX, the combination of gemcitabine (GEM) and docetaxel (DOC), trabectedin (TRA), temozolomide (TEM), pazopanib (PAZ) and olaratumab (OLA). Of these agents GEM/DOC, TRA and TEM were highly effective in the leiomyosarcoma PDOX model, the other agents, including first-line therapy DOX, were ineffective. Thus the leiomyosarcoma PDOX model could precisely distinguish effective and ineffective drugs, demonstrating the potential of the PDOX model for leiomyosarcoma treatment.

Tumor-targeting Salmonella typhimurium A1-R overcomes nab-paclitaxel resistance in a cervical cancer PDOX mouse model.

PURPOSE: Cervical cancer is a recalcitrant disease. To help overcome this problem, we previously established a patient-derived orthotopic xenograft (PDOX) model of cervical cancer. In the previous study, we found the tumor to be resistant to nab-paclitaxal (nab-PTX). We also previously developed the tumor-targeting bacteria Salmonella typhimurium A1-R (S. typhimurium A1-R). The aim of the present study was to investigate the efficacy of S. typhimurium A1-R to overcome nab-PTX resistance in the cervical cancer PDOX model. METHODS: Cervical-cancer tumor fragments were implanted orthotopically into the neck of the uterus of nude mice. The cervical-cancer PDOX models were randomized into the following four groups after the tumor volume reached 60 mm3: G1: untreated group; G2: nab-PTX (i.v., 10 mg/kg, biweekly, 3 weeks); G3: Salmonella typhimurium A1-R (i.v., 5 × 107 CFU/body, weekly, 3 weeks); G4: nab-PTX combined with Salmonella typhimurium A1-R (nab-PTX, 10 mg/kg, i.v., biweekly, 3 weeks; S. typhimurium A1-R, 5 × 107 CFU/body, i.v., weekly, 3 weeks). Each group comprised eight mice. All mice were sacrificed on day 22. Tumor volume was measured on day 0 and day 22. Body weight was measured twice a week. RESULTS: Nab-PTX and Salmonella typhimurium A1-R did not show significant efficacy as monotherapy compared to the control group (P = 0.564 and P = 0.120, respectively). In contrast, nab-PTX combined with Salmonella typhimurium A1-R significantly suppressed tumor growth compared to the untreated control group and nab-PTX group (P < 0.001 and P = 0.026, respectively). CONCLUSIONS: Salmonella typhimurium A1-R has potential future clinical application to overcome drug resistance in cervical cancer.

Tumor-Targeting Salmonella typhimurium A1-R Promotes Tumoricidal CD8+ T Cell Tumor Infiltration and Arrests Growth and Metastasis in a Syngeneic Pancreatic-Cancer Orthotopic Mouse Model.

The present study determined the effect of the tumor-targeting strain Salmonella typhimurium A1-R (S. typhimurium A1-R) on CD8+ tumor-infiltrating lymphocytes (TILs) in a syngeneic pancreatic-cancer orthotopic mouse model. The effect of tumor-targeting S. typhimurium A1-R on CD8+ TILs was determined on the Pan02 murine pancreatic-adenocarcinoma implanted orthotopically in the pancreatic tail of C57BL/6 immunocompromised mice. Three weeks after orthotopic implantation, mice were randomized as follows G1: untreated control group (n = 8); and G2: S. typhimurium A1-R-treatment group (n = 8, 1 × 107 colony forming units [CFU]/body, iv, weekly, 3 weeks). On the 22nd day from initial treatment, all mice were sacrificed and tumors were harvested. The tumor-volume ratio was defined as ratio of tumor volume on the 22nd day relative to the 1st day. The tumor volume ratio was significantly lower in the S. typhimurium A1-R-treated group (G2) (3.0 ± 2.8) than the untreated control (G1) (39.9 ± 30.7, P < 0.01). Hematoxylin and easin (H&E) staining on tumor sections was performed to evaluate tumor destruction which was classified according to the Evans grading system and found to be much greater in the S. typhimurium A1-R-treated mice (G2). Six mice in G1 had peritoneal dissemination, whereas no mice showed peritoneal dissemination in G2 (P < 0.01). Immunohistochemical staining with anti-mouse CD8+ antibody was performed in order to detect TILs determined by calculating the average number of CD8+ cells in three high power fields (200×) in the treated and untreated tumors. The TIL score was significantly higher in G2 (133.5 ± 32.2) than G1 (45.1 ± 19.4, P < 0.001). The present study demonstrates that S. typhimurium A1-R promotes CD8+ T cell infiltration and inhibition of tumor growth and metastasis. J. Cell. Biochem. 119: 634-639, 2018. © 2017 Wiley Periodicals, Inc.

Eribulin regresses a doxorubicin-resistant Ewing's sarcoma with a FUS-ERG fusion and CDKN2A-deletion in a patient-derived orthotopic xenograft (PDOX) nude mouse model.

Ewing's sarcoma is a recalcitrant tumor greatly in need of more effective therapy. The aim of this study was to determine the efficacy of eribulin on a doxorubicin (DOX)-resistant Ewing's sarcoma patient derived orthotopic xenograft (PDOX) model. The Ewing's sarcoma PDOX model was previously established in the right chest wall of nude mice from tumor resected form the patient's right chest wall. In the previous study, the Ewing's sarcoma PDOX was resistant to doxorubicin (DOX) and sensitive to palbociclib and linsitinib. In the present study, the PDOX models were randomized into three groups when the tumor volume reached 60 mm3 : G1, untreated control (n = 6); G2, DOX treated (n = 6), intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, Eribulin treated (n = 6, intravenous (i.v.) injection, weekly for 2 weeks). All mice were sacrificed on day 15. Changes in body weight and tumor volume were assessed two times per week. Tumor weight was measured after sacrifice. DOX did not suppress tumor growth compared to the control group (P = 0.589), consistent with the previous results in the patient and PDOX. Eribulin regressed tumor size significantly compared to G1 and G2 (P = 0.006, P = 0.017) respectively. No significant difference was observed in body weight among any group. Our results demonstrate that eribulin is a promising novel therapeutic agent for Ewing's sarcoma.

Tumor-targeting Salmonella typhimurium A1-R arrests a doxorubicin-resistant PDGFRA-amplified patient-derived orthotopic xenograft mouse model of pleomorphic liposarcoma.

Pleomorphic liposarcoma (PLPS) is a recalcitrant soft-tissue sarcoma (STS) subtype in need of transformative therapy. We have previously established a patient-derived orthotopic xenograft (PDOX) model, of PLPS with PDGFRA amplification, using surgical orthotopic implantation. In the current study, the PLPS PDOX model was randomized into 3 groups of 7 mice each: untreated control; doxorubicin (DOX)-treated; and treated with Salmonella typhimurium A1-R (S. typhimurium A1-R) expressing green fluorescent protein (GFP). Tumor volume and body weight were monitored during the treatment period. The PLPS PDOX was resistant to DOX. In contrast, the PLPS PDOX was highly sensitive to S. typhimurium A1-R. There was no significant body-weight loss among these 3 groups. Fluorescence imaging demonstrated that S. typhimurium A1-R-GFP was very effective to target the PLPS PDOX tumor. The current study demonstrates that a PLPS PDOX, resistant to first-line therapy DOX, was highly sensitive to tumor targeting S. typhimurium A1-R.

Temozolomide regresses a doxorubicin-resistant undifferentiated spindle-cell sarcoma patient-derived orthotopic xenograft (PDOX): precision-oncology nude-mouse model matching the patient with effective therapy.

Undifferentiated spindle-cell sarcoma (USCS) is a recalcitrant cancer, resistant to conventional chemotherapy. A patient with high-grade USCS from a striated muscle was implanted orthotopically in the right biceps femoris muscle of mice to establish a patient-derived orthotopic xenograft (PDOX) model. The PDOX models were randomized into the following groups when tumor volume reached 100 mm3 : G1, control without treatment; G2, doxorubicin (DOX) (3 mg/kg, intraperitoneal [i.p.] injection, weekly, for 2 weeks); G3, temozolomide (TEM) (25 mg/kg, p.o., daily, for 14 days). Tumor size and body weight were measured with calipers and a digital balance twice a week. TEM significantly inhibited tumor volume growth compared to the untreated control and the DOX-treated group on day 14 after treatment initiation: control (G1): 343 ± 78 mm3 ; DOX (G2): 308 ± 31 mm3 , P = 0.272; TEM (G3): 85 ± 21 mm3 , P < 0.0001. TEM significantly regressed the tumor volume compared to day 0 (P = 0.019). There were no animal deaths in any group. The body weight of treated mice was not significantly different in any group. Tumors treated with DOX were comprised of spindle-shaped viable cells without apparent necrosis or inflammatory changes. In contrast, tumors treated with TEM showed extensive tumor necrosis. The present study demonstrates the potential power of matching the patient with an effective drug and saving the patient needless toxicity from ineffective drugs.

Growth of doxorubicin-resistant undifferentiated spindle-cell sarcoma PDOX is arrested by metabolic targeting with recombinant methioninase.

Undifferentiated spindle-cell sarcoma (USCS) is a recalcitrant -cancer in need of individualized therapy. A high-grade USCS from a striated muscle of a patient was grown orthotopically in the right biceps femoris muscle of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. In a previous study, we evaluated the efficacy of standard first-line chemotherapy of doxorubicin (DOX), gemcitabine (GEM) combined with docetaxel (DOC), compared to pazopanib (PAZ), a multi-targeting tyrosine-kinase inhibitor, in an USCS PDOX model. In the present study, mice-bearing the USCS PDOX tumors were randomized into the following groups when tumor volume reached 100 mm3 : G1, untreated control without treatment; G2, DOX (3 mg/kg, intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, L-methionine α-deamino-γ-mercaptomethane lyase (recombinant methioninase [rMETase]) (100 U/mouse, i.p., daily, for 2 weeks). Tumor size and body weight were measured with calipers and a digital balance twice a week. The methionine level of supernatants derived from sonicated tumors was also measured. rMETase inhibited tumor growth, measured by tumor volume, compared to untreated controls and the DOX-treated group on day 14 after initiation of treatment: control (G1): 347.6 ± 88 mm3 ; DOX (G2): 329.5 ± 79 mm3 , P = 0.670; rMETase (G3): 162.6 ± 51 mm3 , P = 0.0003. The mouse body weight of the treated mice was not significantly different from the untreated controls. Tumor L-methionine levels were reduced after the rMETase-treatment compared to untreated control and pre-rMETase treatment. We previously reported efficacy of rMETase against Ewing's sarcoma and melanoma in a PDOX models. These studies suggest clinical development of rMETase, especially in recalcitrant cancers such as sarcoma.

Tumor-targeting Salmonella typhimurium A1-R is a highly effective general therapeutic for undifferentiated soft tissue sarcoma patient-derived orthotopic xenograft nude-mouse models.

Undifferentiated soft tissue sarcoma (USTS) is a recalcitrant and heterogeneous subgroup of soft tissue sarcoma with high risk of metastasis and recurrence. Due to heterogeneity of USTS, there is no reliably effective first-line therapy. We have generated tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R), which previously showed strong efficacy on single patient-derived orthotopic xenograft (PDOX) models of Ewing's sarcoma and follicular dendritic cell sarcoma. In the present study, tumor resected from 4 patients with a biopsy-proven USTS (2 undifferentiated pleomorphic sarcoma [UPS], 1 undifferentiated sarcoma not otherwise specified [NOS] and 1 undifferentiated spindle cell sarcoma [USS]) were grown orthotopically in the biceps femoris muscle of mice to establish PDOX models. One USS model and one UPS model were doxorubicin (DOX) resistant. One UPS and the NOS model were partially sensitive to DOX. DOX is first-line therapy for these diseases. S. typhimurium A1-R arrested tumor growth all 4 models. In addition to arresting tumor growth in each case, S. typhimurium A1-R was significantly more efficacious than DOX in each case, thereby surpassing first-line therapy. These results suggest that S. typhimurium A1-R can be a general therapeutic for USTS and possibly sarcoma in general.

A combination of irinotecan/cisplatinum and irinotecan/temozolomide or tumor-targeting Salmonella typhimurium A1-R arrest doxorubicin- and temozolomide-resistant myxofibrosarcoma in a PDOX mouse model.

Myxofibrosarcoma (MFS) is the most common sarcomas in elderly patients and is either chemo-resistant or recurs with metastasis after chemotherapy. This recalcitrant cancer in need of improved treatment. We have established a patient-derived orthotopic xenograft (PDOX) of MFS. The MFS PDOX model was established in the biceps femoris of nude mice and randomized into 7 groups of 7 mice each: control; doxorubicin (DOX); pazopanib (PAZ); temozolomide (TEM); Irinotecan (IRN); IRN combined with TEM; IRN combined with cisplatinum (CDDP) and Salmonella typhimurium A1-R (S. typhimurium A1-R). Treatment was evaluated by relative tumor volume and relative body weight. The MFS PDOX models were DOX, PAZ, and TEM resistant. IRN combined with TEM and IRN combined with CDDP were most effective on the MFS PDOX. S. typhimurium A1-R arrested the MFS PDOX tumor. There was no significant body weight loss in any group. The present study suggests that the combination of IRN with either TEM or CDDP, and S. typhimurium have clinical potential for MFS.

Combination therapy of tumor-targeting Salmonella typhimurium A1-R and oral recombinant methioninase regresses a BRAF-V600E-negative melanoma.

Melanoma is a recalcitrant cancer. To improve and individualize treatment for this disease, we previously developed a patient-derived orthotopic xenograft (PDOX) model for melanoma. We previously reported the individual efficacy of tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) and recombinant methioninase (rMETase) for melanoma in the PDOX models of this disease. In the present study, we evaluated the efficacy of the combination of S. typhimurium A1-R with orally-administered rMETase (o-rMETase) for BRAF-V600E-negative melanoma in a PDOX model. Three weeks after implantation, 60 PDOX mouse models were randomized into six groups of 10 mice each: untreated control, temozolomide (TEM); o-rMETase; S. typhimurium A1-R; TEM + rMETase, S. typhimurium A1-R + rMETase. All treatments inhibited tumor growth compared to untreated control (TEM: p < 0.0001, rMETase: p < 0.0001, S. typhimurium A1-R: p < 0.0001, TEM + rMETase: p < 0.0001, S. typhimurium A1-R + rMETase: p < 0.0001). The most effective was the combination of S. typhimurium A1-R + o-rMETase which regressed this melanoma PDOX, thereby indicating a new paradigm for treatment of metastatic melanoma.

Metabolic targeting with recombinant methioninase combined with palbociclib regresses a doxorubicin-resistant dedifferentiated liposarcoma.

Liposarcoma is the most common type of soft tissue sarcoma. Among the subtypes of liposarcoma, dedifferentiated liposarcoma (DDLPS) is recalcitrant and has the lowest survival rate. The aim of the present study is to determine the efficacy of metabolic targeting with recombinant methioninase (rMETase) combined with palbociclib (PAL) against a doxorubicin (DOX)-resistant DDLPS in a patient-derived orthotopic xenograft (PDOX) model. A resected tumor from a patient with recurrent high-grade DDLPS in the right retroperitoneum was grown orthotopically in the right retroperitoneum of nude mice to establish a PDOX model. The PDOX models were randomized into the following groups when tumor volume reached 100 mm3: G1, control without treatment; G2, DOX; G3, PAL; G4, recombinant methioninase (rMETase); G5, PAL combined with rMETase. Tumor length and width were measured both pre- and post-treatment. On day 14 after initiation, all treatments significantly inhibited tumor growth compared to the untreated control except DOX. PAL combined with rMETase was significantly more effective than both DOX, rMETase alone, and PAL alone. Combining PAL and rMETase significantly regressed tumor volume on day 14 after initiation of treatment and was the only treatment to do so. The relative body weight on day 14 compared with day 0 did not significantly differ between each treatment group. The results of the present study indicate the powerful combination of rMETase and PAL should be tested clinically against DDLPS in the near future.

Trabectedin arrests a doxorubicin-resistant PDGFRA-activated liposarcoma patient-derived orthotopic xenograft (PDOX) nude mouse model.

BACKGROUND: Pleomorphic liposarcoma (PLPS) is a rare, heterogeneous and an aggressive variant of liposarcoma. Therefore, individualized therapy is urgently needed. Our recent reports suggest that trabectedin (TRAB) is effective against several patient-derived orthotopic xenograft (PDOX) mouse models. Here, we compared the efficacy of first-line therapy, doxorubicin (DOX), and TRAB in a platelet-derived growth factor receptor-α (PDGFRA)-amplified PLPS. METHODS: We used a fresh sample of PLPS tumor derived from a 68-year-old male patient diagnosed with a recurrent PLPS. Subcutaneous implantation of tumor tissue was performed in a nude mouse. After three weeks of implantation, tumor tissues were isolated and cut into small pieces. To match the patient a PDGFRA-amplified PLPS PDOX was created in the biceps femoris of nude mice. Mice were randomized into three groups: Group 1 (G1), control (untreated); Group 2 (G2), DOX-treated; Group 3 (G3), TRAB-treated. Measurement was done twice a week for tumor width, length, and mouse body weight. RESULTS: The PLPS PDOX showed resistance towards DOX. However, TRAB could arrest the PLPS (p < 0.05 compared to control; p < 0.05 compared to DOX) without any significant changes in body-weight. CONCLUSIONS: The data presented here suggest that for the individual patient the PLPS PDOX model could specifically distinguish both effective and ineffective drugs. This is especially crucial for PLPS because effective first-line therapy is harder to establish if it is not individualized.

Combining Tumor-Selective Bacterial Therapy with Salmonella typhimurium A1-R and Cancer Metabolism Targeting with Oral Recombinant Methioninase Regressed an Ewing's Sarcoma in a Patient-Derived Orthotopic Xenograft Model.

BACKGROUND: Ewing's sarcoma (ES) is a recalcitrant disease in need of transformative therapeutics. OBJECTIVES: The aim of this study was to investigate the efficacy of tumor-selective Salmonella typhimurium A1-R combined with tumor metabolism targeting with oral administration of recombinant methioninase (o-rMETase), on an ES patient-derived orthotopic xenograft (PDOX) model. METHODS: The ES PDOX models were previously established in the right chest wall. The ES PDOX models were randomized into 5 groups when the tumor volume reached 80 mm3: G1: untreated control; G2: doxorubicin; G3: S. typhimurium A1-R; G4: o-rMETase; G5: S. typhimurium A1-R combined with o-rMETase. All mice were sacrificed on day 15. Body weight and tumor volume were assessed twice a week. RESULTS: S. typhimurium A1-R and o-rMETase respectively suppressed tumor growth as monotherapies (p = 0.050 and p = 0.032). S. typhimurium A1-R combined with o-rMETase regressed tumor growth significantly compared to untreated group on day 15 (p < 0.032). S. typhimurium A1-R combined with o-rMETase group was significantly more effective than S. typhimurium A1-R or o-rMETase monotherapy (p = 0.032, p = 0.032). CONCLUSIONS: The present results suggest that the combination of S. typhimurium A1-R and o-rMETase has promise to be a transformative therapy for ES.

Analysis of Stroma Labeling During Multiple Passage of a Sarcoma Imageable Patient-Derived Orthotopic Xenograft (iPDOX) in Red Fluorescent Protein Transgenic Nude Mice.

A patient-derived orthotopic xenograft (PDOX) model of undifferentiated pleomorphic sarcoma (UPS) was previously established that acquired red fluorescent protein (RFP)-expressing stroma by growth in an RFP transgenic nude mouse. In the present study, an imageable PDOX model (iPDOX) of UPS was established by orthotopic implantation in the biceps femoris of transgenic RFP nude mice. After the tumors grew to a diameter of 10 mm, they were harvested and the brightest portion of the tumors were subsequently orthotopically transplanted to both RFP and non-colored nude mice. The UPS PDOX tumor was again transplanted to RFP transgenic and non-colored nude mice, and finally a 3rd passage was made in the same manner. Five UPS tumors from each passage in both RFP and non-colored mouse models were harvested. The FV1,000 confocal microscope was used to visualize and quantitate the RFP area of the resected tumors. The average percent fluorescent area in the first passage of RFP mice was 34 ± 22%; in the second passage, 34 ± 20%; and 36 ± 11% in the third passage of RFP transgenic nude mice. The average tumor RFP area in the first passage from RFP mice to non-colored mice was 20 ± 7%; in the second passage, 28 ± 11%; in the third passage was 27 ± 13%. The present results demonstrate the extensive and stable acquisition of stroma by the UPS-tumor growing orthotopically in transgenic RFP nude mice (iPDOX). This model can be used for screening for effective drugs for individual patients and drug discovery. J. Cell. Biochem. 118: 3367-3371, 2017. © 2017 Wiley Periodicals, Inc.

High Efficacy of Pazopanib on an Undifferentiated Spindle-Cell Sarcoma Resistant to First-Line Therapy Is Identified With a Patient-Derived Orthotopic Xenograft (PDOX) Nude Mouse Model.

Undifferentiated spindle-cell sarcoma (USCS) is a recalcitrant -cancer. Our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) nude mouse model with the technique of surgical orthotopic implantation (SOI). In the present study, we evaluated the efficacy of standard first-line chemistry of doxorubicin (DOX), gemcitabine (GEM) combined with docetaxel (DOC), compared to pazopanib (PAZ), a multi-targeting tyrosine-kinase inhibitor, in an USCS PDOX model. A high-grade USCS from a striated muscle of the patients was grown orthotopically in the right biceps femoris muscle of nude mice to establish the PDOX model. The PDOX models were randomized into the following groups when tumor volume reached 100 mm3 : G1, control without treatment; G2, DOX (3 mg/kg, intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, GEM (100 mg/kg, i.p., weekly, for 2 weeks) combined with DOC (20 mg/kg, i.p., once); G4, PAZ (100 mg/kg, p.o., daily, for 14 days). All treatments except DOX significantly inhibited tumor growth compared to untreated control on day 14 after treatment initiation. Tumor sizes were as fallows: control (G1): 332.0 ± 58.7 mm3 ; DOX (G2): 316.9 ± 55.9 mm3 , P = 0.605; GEM + DOC (G3): 228.9 ± 39.8 mm3 , P = 0.001; PAZ (G4): 173.8 ± 23.3 mm3 , P < 0.0001. PAZ showed significantly more efficacy compared to other therapies evaluated: DOX (P < 0.0001), GEM + DOC (P = 0.006). There were no animal deaths in any group and body weight of treated mice was not significantly different in each group. The present results demonstrate that the PDOX model of USCS can identify a promising novel agent with significantly greater efficacy than first-line therapy for this recalcitrant disease. J. Cell. Biochem. 118: 2739-2743, 2017. © 2017 Wiley Periodicals, Inc.

Labeling the Stroma of a Patient-Derived Orthotopic Xenograft (PDOX) Mouse Model of Undifferentiated Pleomorphic Soft-Tissue Sarcoma With Red Fluorescent Protein for Rapid Non-Invasive Imaging for Drug Screening.

Our laboratory pioneered patient-derived orthotopic xenograft (PDOX) mouse models using surgical orthotopic implantation (SOI). PDOX models are patient-like, in contrast to the ectopic subcutaneous-transplant cancer models. In the present study, we demonstrate that an undifferentiated pleomorphic soft-tissue sarcoma (UPS-STS) PDOX model acquired bright RFP-expressing stroma through one passage in red fluorescent protein (RFP) transgenic mice, which upon passage to non-colored nude mice was non-invasively imageable. A PDOX nude mouse model of UPS-STS was established in the biceps femoris of nude mice. After the tumors grew to a diameter of 10 mm, the tumors were subsequently passaged to RFP transgenic mice, and after tumor growth were then passaged to non-transgenic nude mice. Tumors were divided into small fragments and transplanted in the biceps femoris at each passage. The OV100 Small Animal Fluorescence Imaging System and FV1000 laser scanning confocal microscope were used to image RFP fluorescence in the UPS-STS PDOX models. UPS-STS PDOX tumors, previously grown in RFP transgenic nude mice for only one passage, had very bright fluorescence and after passage to non-transgenic nude mice maintained the bright fluorescence and were non-invasively imageable. FV1000 confocal imaging revealed diffusely distributed bright RFP stromal cells in the PDOX tumor, both in RFP transgenic mice and after passage to non-transgenic mice. These results demonstrate a powerful method to make the PDOX UPS-STS model brightly fluorescent for non-invasive imaging, as well as for confocal microscopy of individual stromal cells associated with the tumor. The RFP-labeled UPS PDOX has the potential to rapidly screen for novel effective agents for individual patients, including stroma-targeting drugs, whereby the stromal cells are a visual target. J. Cell. Biochem. 118: 361-365, 2017. © 2016 Wiley Periodicals, Inc.

Tumor-Targeting Salmonella typhimurium A1-R Sensitizes Melanoma With a BRAF-V600E Mutation to Vemurafenib in a Patient-Derived Orthotopic Xenograft (PDOX) Nude Mouse Model.

Previously, a BRAF-V600E-mutant melanoma obtained from the right chest wall of a patient was grown orthotopically in the right chest wall of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. Trametinib (TRA), an MEK inhibitor, caused tumor regression. In contrast, another MEK inhibitor, cobimetinib (COB) could slow but not arrest growth or cause regression of the melanoma PDOX. First-line therapy temozolomide (TEM) could slow but not arrest tumor growth or cause regression. In addition, vemurafenib (VEM) was not effective even though VEM is supposed to target the BRAF-V600E mutation. We also previously demonstrated that tumor-targeting with S. typhimurium A1-R combined with TEM was significantly more effective than either S. typhimurium A1-R alone or TEM alone on the melanoma PDOX with the BRAF-V600E mutation. The present study used this PDOX model of melanoma to test its sensitivity to VEM combined with S. typhimurium A1-R compared to VEM alone and VEM combined with COB. VEM combined with S. typhimurium A1-R was significantly more effective than VEM alone or VEM combined with COB (P = 0.0216) which is currently first line therapy for advanced melanoma with a BRAF-V600E mutation. J. Cell. Biochem. 118: 2314-2319, 2017. © 2017 Wiley Periodicals, Inc.