Patient-Derived Orthotopic Xenograft (PDOX) Models of Melanoma †

Metastatic melanoma is a recalcitrant tumor. Although “targeted” and immune therapies have been highly touted, only relatively few patients have had durable responses. To overcome this problem, our laboratory has established the melanoma patient-derived orthotopic xenograft (PDOX) model with the use of surgical orthotopic implantation (SOI). Promising results have been obtained with regard to identifying effective approved agents and experimental therapeutics, as well as combinations of the two using the melanoma PDOX model.

PD-1/PD-L1 immunotherapy has shown promise with melanoma, but is limited by tumor infiltration of activated T cells [5], and has not increased the survival rate [2].
Stage III and IV melanoma is almost never curable, due to a lack of effective drugs, resistance to immunotherapy and tumor heterogeneity [10]. Chemotherapy and radiotherapy of melanoma are also limited by melanin [11]. Individualized and precision therapy is needed for melanoma.
The present report reviews our laboratory's experience with PDOX models of melanoma, and the ability of the PDOX models to identify effective currently-used-as well as experimental-therapeutics.

S. typhimurium A1-R Was Highly Effective on the Patient-Derived Orthotopic Xenograft (PDOX) Melanoma in Nude Mice
S. typhimurium A1-R, expressing green fluorescent protein (GFP), extensively targeted the tumor, with very few GFP-expressing bacteria found in other organs (i.e., demonstrating high tumor selectivity). S. typhimurium A1-R strongly inhibited the growth of the melanoma (Figure 1). S. typhimurium A1-R, cisplatinum (CDDP), and a combination of S. typhimurium A1-R and CDDP, were all highly effective on the melanoma PDOX ( Figure 2)

PDOX Model of a BRAF-V600E Mutant Melanoma
A BRAF-V600E mutant melanoma PDOX was established. VEM, temozolomide (TEM), trametinib (TRA) and cobimetinib (COB) were all effective against it. TRA treatment caused tumor regression ( Figure 3). The PDOX was expected to be sensitive to VEM, since VEM targets the BRAF-V600E mutation. However, in this case, TRA was much more effective than VEM [55]. This result shows that the BRAF-V600E mutation is probably not a major factor in promoting this melanoma, and that genomic profiling by itself is insufficient to direct therapy. In a subsequent study with this BRAF-V600E mutant melanoma PDOX, TEM combined with S. typhimurium A1-R was significantly more effective than either S. typhimurium A1-R and TEM alone,  [34]. ** p < 0.01, compared with the untreated control group.

PDOX Model of a BRAF-V600E Mutant Melanoma
A BRAF-V600E mutant melanoma PDOX was established. VEM, temozolomide (TEM), trametinib (TRA) and cobimetinib (COB) were all effective against it. TRA treatment caused tumor regression ( Figure 3). The PDOX was expected to be sensitive to VEM, since VEM targets the BRAF-V600E mutation. However, in this case, TRA was much more effective than VEM [55]. This result shows that the BRAF-V600E mutation is probably not a major factor in promoting this melanoma, and that genomic profiling by itself is insufficient to direct therapy.  [34]. ** p < 0.01, compared with the untreated control group.

PDOX Model of a BRAF-V600E Mutant Melanoma
A BRAF-V600E mutant melanoma PDOX was established. VEM, temozolomide (TEM), trametinib (TRA) and cobimetinib (COB) were all effective against it. TRA treatment caused tumor regression ( Figure 3). The PDOX was expected to be sensitive to VEM, since VEM targets the BRAF-V600E mutation. However, in this case, TRA was much more effective than VEM [55]. This result shows that the BRAF-V600E mutation is probably not a major factor in promoting this melanoma, and that genomic profiling by itself is insufficient to direct therapy. In a subsequent study with this BRAF-V600E mutant melanoma PDOX, TEM combined with S. typhimurium A1-R was significantly more effective than either S. typhimurium A1-R and TEM alone, In a subsequent study with this BRAF-V600E mutant melanoma PDOX, TEM combined with S. typhimurium A1-R was significantly more effective than either S. typhimurium A1-R and TEM alone, causing regression of the tumor (Figure 4). Confocal microscopy showed that the S. typhimurium A1-R could directly target the melanoma PDOX and cause tumor necrosis [56].
causing regression of the tumor (Figure 4). Confocal microscopy showed that the S. typhimurium A1-R could directly target the melanoma PDOX and cause tumor necrosis [56]. In a subsequent study, VEM, S. typhimurium A1-R, COB, VEM combined with COB, and VEM combined with S. typhimurium A1-R were all effective against the BRAF-V600E mutant melanoma PDOX, compared to the untreated control. VEM combined with S. typhimurium A1-R was the most effective compared to other therapies ( Figure 5). Tumor necrosis was more extensive in the group treated with VEM combined with S. typhimurium A1-R [9]. In another study, TEM combined with S. typhimurium A1-R, and VEM combined with S. typhimurium A1-R, were significantly more effective than S. typhimurium A1-R alone on the BRAF-V600E mutant melanoma PDOX ( Figure 6). Both VEM and TEM significantly increased the tumor targeting of S. typhimurium A1-R, compared to S. typhimurium A1-R alone, as observed by highresolution confocal microscopy ( Figure 7A,B). These results suggested that S. typhimurium A1-R increases the efficacy of chemotherapy, and chemotherapy increases the tumor targeting of S. typhimurium A1-R in the melanoma PDOX model [57]. In a subsequent study, VEM, S. typhimurium A1-R, COB, VEM combined with COB, and VEM combined with S. typhimurium A1-R were all effective against the BRAF-V600E mutant melanoma PDOX, compared to the untreated control. VEM combined with S. typhimurium A1-R was the most effective compared to other therapies ( Figure 5). Tumor necrosis was more extensive in the group treated with VEM combined with S. typhimurium A1-R [9]. causing regression of the tumor (Figure 4). Confocal microscopy showed that the S. typhimurium A1-R could directly target the melanoma PDOX and cause tumor necrosis [56]. In a subsequent study, VEM, S. typhimurium A1-R, COB, VEM combined with COB, and VEM combined with S. typhimurium A1-R were all effective against the BRAF-V600E mutant melanoma PDOX, compared to the untreated control. VEM combined with S. typhimurium A1-R was the most effective compared to other therapies ( Figure 5). Tumor necrosis was more extensive in the group treated with VEM combined with S. typhimurium A1-R [9]. In another study, TEM combined with S. typhimurium A1-R, and VEM combined with S. typhimurium A1-R, were significantly more effective than S. typhimurium A1-R alone on the BRAF-V600E mutant melanoma PDOX ( Figure 6). Both VEM and TEM significantly increased the tumor targeting of S. typhimurium A1-R, compared to S. typhimurium A1-R alone, as observed by highresolution confocal microscopy ( Figure 7A,B). These results suggested that S. typhimurium A1-R increases the efficacy of chemotherapy, and chemotherapy increases the tumor targeting of S. typhimurium A1-R in the melanoma PDOX model [57]. In another study, TEM combined with S. typhimurium A1-R, and VEM combined with S. typhimurium A1-R, were significantly more effective than S. typhimurium A1-R alone on the BRAF-V600E mutant melanoma PDOX ( Figure 6). Both VEM and TEM significantly increased the tumor targeting of S. typhimurium A1-R, compared to S. typhimurium A1-R alone, as observed by high-resolution confocal microscopy ( Figure 7A,B). These results suggested that S. typhimurium A1-R increases the efficacy of chemotherapy, and chemotherapy increases the tumor targeting of S. typhimurium A1-R in the melanoma PDOX model [57]. Methionine dependence is a general metabolic defect in cancer. It has been demonstrated that methionine starvation induces a tumor-selective S/G2-phase cell-cycle arrest of tumor cells [58][59][60][61]. Methionine dependence is due to the excess use of methionine in aberrant transmethylation reactions, termed the Hoffman effect, and is analogous to the Warburg effect for glucose in cancer [62][63][64][65][66][67]. The excessive and aberrant use of methionine in cancer is strongly observed in [ 11 C]-methionine PET imaging, where the high uptake of [ 11 C]-methionine results in a very strong and selective tumor signal compared with normal tissue background. [ 11 C]-methionine is superior to [ 18 C]fluorodeoxyglucose (FDG) for PET imaging, suggesting methionine dependence is more tumorspecific than glucose dependence [68,69]. A purified methionine-cleaving enzyme, methioninase (METase), from Pseudomonas putida, has been found previously to be an effective antitumor agent in vitro as well as in vivo [70][71][72][73]. For the large-scale production of METase, the gene from P. putida has been cloned in Escherichia coli and a purification protocol for recombinant methioninase (rMETase) has been established with high purity and low endotoxin release [74][75][76][77]. Methionine dependence is a general metabolic defect in cancer. It has been demonstrated that methionine starvation induces a tumor-selective S/G 2 -phase cell-cycle arrest of tumor cells [58][59][60][61]. Methionine dependence is due to the excess use of methionine in aberrant transmethylation reactions, termed the Hoffman effect, and is analogous to the Warburg effect for glucose in cancer [62][63][64][65][66][67]. The excessive and aberrant use of methionine in cancer is strongly observed in [ 11 C]-methionine PET imaging, where the high uptake of [ 11 C]-methionine results in a very strong and selective tumor signal compared with normal tissue background. [ 11 C]-methionine is superior to [ 18 C]-fluorodeoxyglucose (FDG) for PET imaging, suggesting methionine dependence is more tumor-specific than glucose dependence [68,69]. A purified methionine-cleaving enzyme, methioninase (METase), from Pseudomonas putida, has been found previously to be an effective antitumor agent in vitro as well as in vivo [70][71][72][73]. For the large-scale production of METase, the gene from P. putida has been cloned in Escherichia coli and a purification protocol for recombinant methioninase (rMETase) has been established with high purity and low endotoxin release [74][75][76][77]. Methionine dependence is a general metabolic defect in cancer. It has been demonstrated that methionine starvation induces a tumor-selective S/G2-phase cell-cycle arrest of tumor cells [58][59][60][61]. Methionine dependence is due to the excess use of methionine in aberrant transmethylation reactions, termed the Hoffman effect, and is analogous to the Warburg effect for glucose in cancer [62][63][64][65][66][67]. The excessive and aberrant use of methionine in cancer is strongly observed in [ 11 C]-methionine PET imaging, where the high uptake of [ 11 C]-methionine results in a very strong and selective tumor signal compared with normal tissue background. [ 11 C]-methionine is superior to [ 18 C]fluorodeoxyglucose (FDG) for PET imaging, suggesting methionine dependence is more tumorspecific than glucose dependence [68,69]. A purified methionine-cleaving enzyme, methioninase (METase), from Pseudomonas putida, has been found previously to be an effective antitumor agent in vitro as well as in vivo [70][71][72][73]. For the large-scale production of METase, the gene from P. putida has been cloned in Escherichia coli and a purification protocol for recombinant methioninase (rMETase) has been established with high purity and low endotoxin release [74][75][76][77].  The combination therapy of TEM and rMETase had significantly better efficacy than either therapy alone on the BRAF-V600E mutant melanoma PDOX (Figure 8). Post-treatment L-methionine levels in tumors treated with rMETase alone, or along with TEM, were significantly decreased compared to untreated controls (data not shown). These results showed that this melanoma is methionine dependent, and rMETase thereby suppresses the melanoma PDOX [77].  The combination therapy of TEM and rMETase had significantly better efficacy than either therapy alone on the BRAF-V600E mutant melanoma PDOX (Figure 8). Post-treatment L-methionine levels in tumors treated with rMETase alone, or along with TEM, were significantly decreased compared to untreated controls (data not shown). These results showed that this melanoma is methionine dependent, and rMETase thereby suppresses the melanoma PDOX [77]. The combination therapy of TEM and rMETase had significantly better efficacy than either therapy alone on the BRAF-V600E mutant melanoma PDOX (Figure 8). Post-treatment L-methionine levels in tumors treated with rMETase alone, or along with TEM, were significantly decreased compared to untreated controls (data not shown). These results showed that this melanoma is methionine dependent, and rMETase thereby suppresses the melanoma PDOX [77].  This review indicates that the melanoma PDOX is a promising-although still-developingtechnology, able to identify effective therapy for patients, both approved and experimental. Future studies will investigate further advantages of the melanoma PDOX model. Please see references [78,79] for reviews of melanoma PDX models. Future studies will address molecular changes in the treated melanoma PDOX models described in the present report.

Mice
Athymic (nu/nu) nude mice (AntiCancer Inc., San Diego, CA, USA) were used in these studies in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals under Assurance Number A3873-1. Animals were anesthetized with a ketamine mixture via subcutaneous injection of a 0.02 mL solution of 20 mg/kg ketamine, 15.2 mg/kg xylazine and 0.48 mg/kg acepromazine maleate for all surgeries [9,[55][56][57]77].

Patient-Derived Tumors
The PDOX models from the University of California Los Angeles (UCLA) were established from a 75-year-old female patient with a melanoma of the right chest wall. The melanoma had a BRAF-V600E mutation. Tumor resection was performed in the Department of Surgery, UCLA. The tumor was provided for PDOX establishment after written informed consent was provided by the patient, and after approval was granted by the Institutional Review Board (IRB) [55]. Another patient melanoma was obtained from a patient at UCSD under IRB approval and informed patient consent [34].

Establishment of PDOX Models of Melanoma by Surgical Orthotopic Implantation (SOI)
Resected melanoma tissue was immediately transported to AntiCancer Inc. on ice. The BRAF-V600E mutant melanoma tumor fragments (3 mm 3 ) were transplanted to the chest wall of nude mice to mimic the site from which they were resected from the patient [9,[55][56][57]77]. The melanoma from UCSD was directly implanted subdermally and passaged in the back skin of nude mice [34]. All surgeries were performed under ketamine anesthesia.

Tumor Histology
The original tumor tissue and PDOX tumor tissue were fixed in 10% formalin. The fixed tumors were embedded in paraffin and then sectioned and stained. Standard bright-light microscopy was used for histopathological analysis [55].

Intratumor L-Methionine Levels
After the completion of rMETase treatment, each tumor was sonicated for 30 s on ice and centrifuged at 12,000 rpm for 10 min. Supernatants were collected and protein levels were measured using the Coomassie Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). L-methionine levels were determined using a high-pressure liquid chromatography (HPLC) procedure we developed previously [81,82]. Methionine levels were normalized to tumor protein by standard procedures.

Conflicts of Interest:
The author declares no conflict of interest.