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Supplementary Movie from Time-Course Imaging of Therapeutic Functional Tumor Vascular Normalization by Antiangiogenic Agents

PDF file - 291K, Figure S1. A1-R SIINF preferentially accumulates in established B16-OVA tumors. Figure S2. A1-R SIINF induces systemic SIINF-specific CD8+ T cell proliferation. Figure S3. Weekly A1-R SIINF treatment leads to rejection of established B16-OVA tumors. Figure S4. The majority of intratumoral SIINF-specific CD8+ T cells still express a high level of PD-1 following A1-R OVA treatment.

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Supplementary Methods, Figures 1-3, Movie Legend from Time-Course Imaging of Therapeutic Functional Tumor Vascular Normalization by Antiangiogenic Agents

Supplementary Movie S1- MOV file 6383K, Time-lapse imaging of FUCCI-expressing CD133+ cells treated with OBP-301 or control on monolayer culture. Images were acquired every 20 min. The playback speed is 10, 800 x real time. Total imaging time = 48 hours. The cells in G0/ G1, S, or G2/M phases appear red, yellow, or green, respectively

Purpose: Targeting the protein neddylation pathway has become an attractive anticancer strategy; however, the role of death receptor–mediated extrinsic apoptosis during treatment remained to be determined.Experimental Design: The activation of extrinsic apoptosis and its role in MLN4924 treatment of human esophageal squamous cell carcinoma (ESCC) were evaluated both in vitro and in vivo. The expression of the components of extrinsic apoptotic pathway was determined by immunoblotting analysis and downregulated by siRNA silencing for mechanistic studies.Results: Pharmaceutical or genetic inactivation of neddylation pathway induced death receptor 5 (DR5)–mediated apoptosis and led to the suppression of ESCC in murine models. Mechanistically, neddylation inhibition stabilized activating transcription factor 4 (ATF4), a Cullin-Ring E3 ubiquitin ligases (CRL) substrate. Transcription factor CHOP was subsequently transactivated by ATF4 and further induced the expression of DR5 to activate caspase-8 and induce extrinsic apoptosis. Moreover, the entire neddylation pathway was hyperactivated in ESCC and was negatively associated with patient overall survival.Conclusions: Our findings highlight a critical role of ATF4–CHOP–DR5 axis-mediated extrinsic apoptosis in neddylation-targeted cancer therapy and support the clinical investigation of neddylation inhibitors (e.g., MLN4924) for the treatment of ESCC, a currently treatment-resistant disease with neddylation hyperactivation. Clin Cancer Res; 22(16); 4145–57. ©2016 AACR.

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PDF file 1712K, Supplementary Methods Fig. S1. OBP-301 efficiently kills CD133+ cells from MKN7 cells resistant to conventional therapies. Fig. S2. CD133+ CS-like cells can produce CD133+ CS-like cells and CD133- non-CS-like cells. Fig. S3. CD133+ MKN7 cells produce larger colonies than CD133- MKN7 cells in vitro. Fig. S4. CD133+ cells have higher tumorigenicity in nude mice than CD133− cells. Fig. S5. Flow cytometry analysis shows that OBP-301 induces a significant increase in the percentage of both CD133+ CS-like and CD133- non-CS-like cells in S phase. Fig. S6. Expression of CD133 mRNA is associated with the population of CD133+ cells. Fig. S7. OBP-301 reduces the expression of CD133 mRNA. Fig. S8. CD133+ cells show higher expressions of p53, p21 and p27 proteins compared with CD133− cells. Fig. S9. Adenoviral E1A alters cell-cycle related proteins. Fig. S10. Experimental setup in vivo. Fig. S11. Experimental setup for combination therapy of OBP-301 plus cisplatin or paclitaxel in vitro. Fig. S12. The combination therapy of OBP-301 and chemotherapy is more effective than OBP-301 monotherapy on monolayer culture. Fig. S13. OBP-301 reduces the potentiality of stemness in CD133 positive cells. Fig. S14. The combination therapy of OBP-301 and chemotherapy is more effective against larger tumors. Fig. S15. A scheme for oncolytic adenovirus killing quiescent human cancer cells

Purpose: Because chemoradiotherapy selectively targets proliferating cancer cells, quiescent cancer stem–like cells are resistant. Mobilization of the cell cycle in quiescent leukemia stem cells sensitizes them to cell death signals. However, it is unclear that mobilization of the cell cycle can eliminate quiescent cancer stem–like cells in solid cancers. Thus, we explored the use of a genetically-engineered telomerase-specific oncolytic adenovirus, OBP-301, to mobilize the cell cycle and kill quiescent cancer stem–like cells.Experimental Design: We established CD133+ cancer stem–like cells from human gastric cancer MKN45 and MKN7 cells. We investigated the efficacy of OBP-301 against quiescent cancer stem–like cells. We visualized the treatment dynamics of OBP-301 killing of quiescent cancer stem–like cells in dormant tumor spheres and xenografts using a fluorescent ubiquitination cell-cycle indicator (FUCCI).Results: CD133+ gastric cancer cells had stemness properties. OBP-301 efficiently killed CD133+ cancer stem–like cells resistant to chemoradiotherapy. OBP-301 induced cell-cycle mobilization from G0–G1 to S/G2/M phases and subsequent cell death in quiescent CD133+ cancer stem–like cells by mobilizing cell-cycle–related proteins. FUCCI enabled visualization of quiescent CD133+ cancer stem–like cells and proliferating CD133− non–cancer stem–like cells. Three-dimensional visualization of the cell-cycle behavior in tumor spheres showed that CD133+ cancer stem–like cells maintained stemness by remaining in G0–G1 phase. We showed that OBP-301 mobilized quiescent cancer stem–like cells in tumor spheres and xenografts into S/G2/M phases where they lost viability and cancer stem–like cell properties and became chemosensitive.Conclusion: Oncolytic adenoviral infection is an effective mechanism of cancer cell killing in solid cancer and can be a new therapeutic paradigm to eliminate quiescent cancer stem–like cells. Clin Cancer Res; 19(23); 6495–505. ©2013 AACR.

Supplementary Table S1. Clinicopathologic parameters according to the expression of NAE1, UBA3 and UBC12. Supplementary Table S2. Clinicopathologic parameters according to the expression of global neddylation (global NEDD 8 conjugation).

Supplementary Figure S1. Expression of the global neddylation protein, NEDD8-activating enzyme E1 and NEDD8-conjugating enzyme E2 in ESCC tissues Supplementary Figure S2. Expression of death-receptor family and Bcl-2 family members Supplementary Figure S3. MLN4924 acted on DR5-mediated apoptosis independently of Trail. Kyse450 and EC1 cells were transfected (48 hours) with control or Trail siRNA, treated with 0.6 μM MLN4924 (48 hours). Supplementary Figure S4. MLN4924 inhibited the polyubiquitination modification of ATF4. A, MLN4924 increased the mRNA level of CHOP while little effect on ATF4 mRNA level. Supplementary Figure S5. A, Knockdown efficiency of ATF4. Kyse450 and EC1 cells were transfected with control or ATF4 siRNA for 24 hours and then treated with 0.6 μM MLN4924 for 48 hours Supplementary Figure S6. Genetic inactivation of neddylation pathway via NAE1/UBA3 siRNA silencing recapitulates MLN4924-induced cellular responses in ESCC cells. Supplementary Figure S7. No body weight loss occurred during treatment. Body weight of the animals was measured and recorded during the treatment period.

Supplementary Table 2 from Salmonella Promoters Preferentially Activated Inside Tumors

To noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment and drug response at the cellular level in live animals in real time, we developed a new imageable three-color animal model. The model consists of green fluorescent protein (GFP)–expressing mice transplanted with dual-color cancer cells labeled with GFP in the nucleus and red fluorescent protein in the cytoplasm. The Olympus IV100 Laser Scanning Microscope, with ultra-narrow microscope objectives (“stick objectives”), is used for three-color whole-body imaging of the two-color cancer cells interacting with the GFP-expressing stromal cells. In this model, drug response of both cancer and stromal cells in the intact live animal is also imaged in real time. Various in vivo phenomena of tumor-host interaction and cellular dynamics were imaged, including mitotic and apoptotic tumor cells, stromal cells interacting with the tumor cells, tumor vasculature, and tumor blood flow. This new model system enables the first cellular and subcellular images of unperturbed tumors in the live intact animal. New visible real-time targets for novel anticancer agents are provided in this model, including the color-coded interacting cancer and stromal cells, tumor vasculature, and blood flow. This imageable model should lead to many new insights of in vivo cancer cell biology and to novel drug discovery. [Cancer Res 2007;67(11):5195–200]

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Supplementary Figure 2 from KRas Induces a Src/PEAK1/ErbB2 Kinase Amplification Loop That Drives Metastatic Growth and Therapy Resistance in Pancreatic Cancer

Supplementary Movie 1 from Real-time Imaging of Tumor-Cell Shedding and Trafficking in Lymphatic Channels

Supplementary Appendix 2A from High-Throughput Screening for Salmonella Avirulent Mutants That Retain Targeting of Solid Tumors

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Supplementary Movie 1 from Whole-Body Subcellular Multicolor Imaging of Tumor-Host Interaction and Drug Response in Real Time

Supplementary Movie 2 from Development of Real-time Subcellular Dynamic Multicolor Imaging of Cancer-Cell Trafficking in Live Mice with a Variable-Magnification Whole-Mouse Imaging System

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Supplementary Movie 3 from Development of Real-time Subcellular Dynamic Multicolor Imaging of Cancer-Cell Trafficking in Live Mice with a Variable-Magnification Whole-Mouse Imaging System

Salmonella enterica and avirulent derivatives prefer solid tumors over normal tissue in animal models. The identification of endogenous Salmonella promoters that are preferentially activated in tumors could further our understanding of this phenomenon. Toward this goal, a random library of S. enterica typhimurium 14028 genomic DNA was cloned upstream of a promoterless gene encoding the green fluorescent protein (GFP) TurboGFP. A population of Salmonella containing this library was injected i.v. into tumor-free nude mice and into human PC3 prostate tumors growing subcutaneously in nude mice. After 2 days, fluorescence-activated cell sorting was used to enrich for bacterial clones expressing GFP from spleens or tumors. The resulting libraries were hybridized to an oligonucleotide tiling array of the Salmonella genome. Eighty-six intergenic regions were found to be enriched in tumor samples but not in spleen. Twenty of these candidate promoters were also detected in the sequences of 100 random clones from a library enriched for expression in bacteria growing in tumors. Three candidate promoter clones were individually tested in vivo, and enhanced GFP expression in bacteria growing in tumor relative to spleen was confirmed. Two of the three clones (pflE and ansB promoter regions) are known to be induced in hypoxic conditions that pertain to many tumors. For many of the other candidate promoters preferentially induced in bacteria growing in tumors, regulatory mechanisms may not be related to hypoxia. The expression of therapeutics in Salmonella under the regulation of one or more promoters that are activated preferentially in tumors has the potential to improve the targeting of drug delivery. [Cancer Res 2008;68(12):4827–32]

With the use of dual-color fluorescent cells and a highly sensitive whole-mouse imaging system with both macro-optics and micro-optics, we report here the development of subcellular real-time imaging of cancer cell trafficking in live mice. To observe cytoplasmic and nuclear dynamics in the living mouse, tumor cells were labeled in the nucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with an Olympus OV100 whole-mouse imaging system with a sensitive CCD camera and five objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. With the dual-color cancer cells and the highly sensitive whole-mouse imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time. This imaging technology will enable further understanding of the critical steps of metastasis and provide visible targets for antimetastasis drug development. (Cancer Res 2006; 66(8): 4208-14)

Supplementary Table 5 from Salmonella Promoters Preferentially Activated Inside Tumors

Early biomarkers and effective therapeutic strategies are desperately needed to treat pancreatic ductal adenocarcinoma (PDAC), which has a dismal 5-year patient survival rate. Here, we report that the novel tyrosine kinase PEAK1 is upregulated in human malignancies, including human PDACs and pancreatic intraepithelial neoplasia (PanIN). Oncogenic KRas induced a PEAK1-dependent kinase amplification loop between Src, PEAK1, and ErbB2 to drive PDAC tumor growth and metastasis in vivo. Surprisingly, blockade of ErbB2 expression increased Src-dependent PEAK1 expression, PEAK1-dependent Src activation, and tumor growth in vivo, suggesting a mechanism for the observed resistance of patients with PDACs to therapeutic intervention. Importantly, PEAK1 inactivation sensitized PDAC cells to trastuzumab and gemcitabine therapy. Our findings, therefore, suggest that PEAK1 is a novel biomarker, critical signaling hub, and new therapeutic target in PDACs. Cancer Res; 72(10); 2554–64. ©2012 AACR.

Supplementary Table 1 from Salmonella Promoters Preferentially Activated Inside Tumors

Supplementary Movie 1 from Development of Real-time Subcellular Dynamic Multicolor Imaging of Cancer-Cell Trafficking in Live Mice with a Variable-Magnification Whole-Mouse Imaging System

Supplementary Table 4 from Salmonella Promoters Preferentially Activated Inside Tumors

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Supplementary Table 3 from Salmonella Promoters Preferentially Activated Inside Tumors

Supplementary Figure 2 from Glioma Tumor Stem-Like Cells Promote Tumor Angiogenesis and Vasculogenesis via Vascular Endothelial Growth Factor and Stromal-Derived Factor 1

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Supplementary Figure 1 from Glioma Tumor Stem-Like Cells Promote Tumor Angiogenesis and Vasculogenesis via Vascular Endothelial Growth Factor and Stromal-Derived Factor 1

Supplementary Movie 3 from Real-time Imaging of Tumor-Cell Shedding and Trafficking in Lymphatic Channels

Supplementary Figure Legends 1-2 from Glioma Tumor Stem-Like Cells Promote Tumor Angiogenesis and Vasculogenesis via Vascular Endothelial Growth Factor and Stromal-Derived Factor 1

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Supplementary Movie 2 from Whole-Body Subcellular Multicolor Imaging of Tumor-Host Interaction and Drug Response in Real Time

Background/Aim: Methionine addiction is a general and fundamental hallmark of cancer cells, termed the Hoffman effect. Previously Vanhamme and Szpirer showed that methionine addiction could be induced by transfection of the activated HRAS1 gene to a normal cell line. In the present study, we investigated the role of the c-MYC oncogene in methionine addiction of cancer, by comparison of c-Myc expression and malignancy of methionine-addicted osteosarcoma cells and rare methionine-independent revertants, derived from the methionine-addicted cells. Materials and Methods: Methionine-independent revertant 143B osteosarcoma cells (143B-R) were derived from methionine-addicted parental 143B osteosarcoma cells (143B-P), by continuous culture in medium depleted of methionine by recombinant methioninase. To compare in vitro malignancy of methionine-addicted parental cells and methionine-independent revertant cells, the following experiments were performed: for 143B-P and 143B-R cells, cell proliferation capacity was measured with a cell-counting assay, and colony-formation capacity was determined on plastic and in soft agar, all in methionine-containing Dulbecco’s Modified Eagle’s Medium (DMEM). Tumor growth was measured in orthotopic xenograft nude-mouse models, to compare in vivo malignancy of 143B-P and 143B-R cells. c-MYC expression was examined with western immunoblotting and compared in 143B-P and 143B-R cells. Results: 143B-R cells had reduced cell proliferation capacity, compared to 143B-P cells, in methionine-containing medium (p=0.003). 143B-R cells had reduced colony formation capacity on plastic (p=0.003) and in soft agar, compared to 143B-P cells in methionine-containing medium. 143B-R cells had reduced tumor growth in orthotopic xenograft nude-mouse models, compared to 143B-P cells, (p=0.002). These results demonstrate that 143B-R methionine-independent revertant cells lost malignancy. Expression of c-MYC was reduced in 143B-R methionine-independent revertant osteosarcoma cells, compared to 143B-P cells, (p=0.0007). Conclusion: The present study demonstrated that c-MYC expression is linked to malignancy and methionine addiction of cancer cells. The present study on c-MYC, and the previous study on HRAS1, suggest that oncogenes may play a role in methionine addiction, which is a hallmark of all cancers, as well as in malignancy.

Mucins (MUC1–MUC24) are a family of glycoproteins involved in cell signaling and barrier protection. They have been implicated in the progression of numerous malignancies including gastric, pancreatic, ovarian, breast, and lung cancer. Mucins have also been extensively studied with respect to colorectal cancer. They have been found to have diverse expression profiles amongst the normal colon, benign hyperplastic polyps, pre-malignant polyps, and colon cancers. Those expressed in the normal colon include MUC2, MUC3, MUC4, MUC11, MUC12, MUC13, MUC15 (at low levels), and MUC21. Whereas MUC5, MUC6, MUC16, and MUC20 are absent from the normal colon and are expressed in colorectal cancers. MUC1, MUC2, MUC4, MUC5AC, and MUC6 are currently the most widely covered in the literature regarding their role in the progression from normal colonic tissue to cancer.

Osteosarcoma is the most common bone sarcoma. Although surgery and chemotherapy are initially effective, the 5-year survival is approximately 60% to 80%, and has not improved over three decades. We have previously shown that methionine restriction (MR) induced by oral recombinant methioninase (o-rMETase), is effective against osteosarcoma in patient-derived orthotopic xenograft (PDOX) nude-mouse models. In the present report, the efficacy of the combination of oral o-rMETase and methotrexate (MTX) was examined in an osteosarcoma PDOX mouse model.An osteosarcoma-PDOX model was previously established by implanting tumor fragments into the proximal tibia of nude mice. The osteosarcoma PDOX models were randomized into four groups: control; o-rMETase alone; MTX alone; combination of o-rMETase and MTX. The mice were sacrificed after 4 weeks of treatment.The combination of o-rMETase and MTX showed significantly higher efficacy compared to the control group (p=0.04). The combination also showed significantly higher efficacy compared to MTX alone (p=0.04). No significant efficacy of o-rMETase alone or MTX alone compared to control was shown (p=0.21, 1.00, respectively). Only the combination of o-rMETase and MTX reduced the cancer-cell density in the osteosarcoma tumor.rMETase converted an osteosarcoma PDOX from MTX-resistant to MTX-sensitive and thereby shows future clinical potential.

Attempts to selectively starve cancers in the clinic have been made at least since the time of Warburg beginning 100 years ago. Calorie-restriction or low-carbohydrate diets have had limited success with cancer patients. Methionine restriction is another strategy to selectively starve cancer cells, since cancers are addicted to methionine, unlike normal cells. Methionine addiction of cancer is termed the Hoffman effect. Numerous preclinical studies over the past half century have shown methionine restriction to be highly effective against all major cancer types and synergistic with chemotherapy. Low-methionine medical diets can be effective in lowering methionine and have shown some clinical promise, but they are not palatable and thereby not sustainable. However, selectively choosing among plant-based foods allows a variety of low-methionine diets that are sustainable. Our laboratory has developed a methioninase that can be administered orally as a supplement and has resulted in anecdotal positive results in patients with advanced cancer, including hormone-independent prostate cancer, and other recalcitrant cancers. The question is whether methionine restriction through a low-methionine diet, or even greater methionine restriction with methioninase in combination with a low-methionine diet, is ready for prime time in the clinic, especially in combination with other synergistic therapy. The question will hopefully be answered in the near future, especially for advanced cancer patients who have failed all standard therapy.

Background/Aim: Although the 5-year survival rate for localized prostate cancer is nearly 100%, prognosis for patients with metastases, of which the bone is the most common site, is poor. In order to evaluate efficacy of treatments against metastatic prostate cancer, experimental tibia-bone-metastasis mouse models of prostate cancer have been previously established. In the present study, we used a novel procedure for establishment of an experimental tibia-bone metastasis mouse model, with human PC-3 prostate cancer expressing green fluorescent protein (GFP), that more closely matches prostate cancer growing in the bone. Materials and Methods: PC-3 human prostate cancer cells, labeled with GFP, were initially subcutaneously injected into the flank of five male nude mice to obtain tumor tissues. Once the tumor tissue grew larger than 10 mm in diameter, the tumor tissue was harvested and minced into fragments of 1 mm(3). A 1-mm hole was made in the proximal left tibia of eight male nude mice, using the tip of a 5-mm blade, and a tumor fragment was implanted into the hole for an exact fit. Tumor size was measured once a week, by non-invasive imaging of GFP fluorescence. The mice were sacrificed four weeks after tumor implantation. Results: Tumors grew in 8 out of 8 mice (100%). All tumors were non-invasively detectable with GFP fluorescence, through the skin. Increased tumor growth in the tibia was observed every week. Conclusion: The establishment in the tibia of the novel experimental bone-metastatic mouse model of human prostate cancer enables facile screening, in a clinically-relevant system, of improved therapeutics for this recalcitrant disease.

Background/Aim: Obesity is a major risk factor for colorectal cancer. The MC38 mouse colon-cancer cell line is a versatile syngeneic model of colon cancer in C57BL/6 mice. In the present study, the influence of a high-fat diet (HFD) on the growth of the MC38 mouse colon-cancer cell line was examined in an orthotopic-transplantation syngeneic model in C57BL/6 mice. Materials and Methods: Five 6-week-old C57BL/6 male mice were fed a control diet (CD, 6.5% fat) or HFD (34.3% fat) for eight weeks. Then, a 2 mm(3) fragment of a subcutaneous MC38 tumor was attached to the surface of the cecum of C57BL/6 mice with a single stitch using a 7-0 suture to establish an orthotopic-transplantation model. Each group continued their initial diet for 17 days. Results: The HFD group had more than twice the tumor volume and tumor weight than the CD group (p=0.021 and p=0.014, respectively). Conclusion: HFD-induced obesity strongly increased MC38 colon-cancer progression in a C57BL/6 orthotopic-transplantation mouse model. The present study emphasizes the detrimental effect of obesity on colon-cancer progression.