Abstract
Cancer patients infected with various bacteria were reported, for at least two centuries, to have spontaneous remission. W.B. Coley, of what is now the Memorial Sloan-Kettering Cancer Center, pioneered bacterial therapy of cancer in the clinic with considerable success beginning in the late nineteenth century. After Coley died in 1936, bacterial therapy of cancer essentially ended. Currently there is much excitement in developing bacterial therapy for treating cancer using either obligate or facultative anaerobic bacteria. This chapter will demonstrate the potential and strategy of Salmonella typhimurium A1-R, an engineered tumor-targeting variant for the systemic treatment of metastatic cancer. A new concept using Salmonella typhimurium A1-R for cell cycle “decoy” chemotherapy of metastatic cancer is also described.
Key words
The Outsider is … a person who lives on the edge, challenges culture values and ‘stands for truth’. ” Colin Wilson: The Outsider, Tarcher/Penguin, New York, 1982.
1 Introduction
Cancer patients have been recorded going into remission after a bacterial infection [1]. For example, the German physician Busch in 1867 reported that a malignant cancer went into remission when the patient became infected with erysipelas, now known as Streptococcus pyogenes. Then, in 1888, Bruns treated a cancer patient with S. pyogenes, and the tumor regressed [1, 2].
William B. Coley at New York Cancer Hospital, the precursor of Sloan-Kettering Memorial Cancer Center, treated cancer patients with S. pyogenes beginning in the very late nineteenth century. Coley first treated a patient with head and neck cancer and the tumor regressed. Coley injected many cancer patients with S. pyogenes and often had good results. Coley then mixed killed S. pyogenes with a second killed organism, Serratia marcescens. The mixture became known as Coley’s Toxins.
Hoption Cann et al. [3] compared Coley’s results to results with modern conventional treatments. The 10-year survival rates of patients were compared with the Surveillance Epidemiology End Result Cancer Registry [4]. Hoption Cann’s meta-analysis found that patients receiving current conventional therapies did not survive longer than patients who received bacterial treatment by Coley over 100 years ago.
Tumor characteristics, which are barriers to standard therapy such as a poor vascularity and hypoxia, are facilitators of bacterial therapy [2].
Bifidobacterium [5] and Clostridium [6] are anaerobic bacteria which replicate only in necrotic areas of tumors. Anaerobic bacteria cannot grow in viable tumor tissue, which limits their efficacy, must be used in combination with chemotherapy in order to be effective and may be limited into intra-tumor (i.t.) administration [2, 7]. Yazawa et al. observed that Bifidobacterium longum was effective against a chemically-induced tumor [5]. Clostridium novyi with its lethal toxin removed (no toxin [NT]), was generated. C. novyi-NT spores germinated within the avascular regions of tumors in mice and killed surrounding viable tumor cells after intravenous (i.v.) injection. When C. novyi-NT spores were administered in combination with chemotherapy, hemorrhagic necrosis of tumors developed and the tumors regressed [6].
The disadvantage of the obligate anaerobes described above is that they do not grow in viable regions of tumors due to high oxygen tension, and that is why they may have to be administered i.t. Salmonella typhimurium (S. typhimurium), is a facultative anaerobe which has important advantages, compared to an obligate anaerobe. A facultative anaerobe can grow in the oxic viable region of tumors as well as necrotic regions, as opposed to an obligate anaerobe which only can grow in the necrotic regions of tumors [8]. Wild-type S. typhimurium administered colonized melanoma tumors at 109/g but killed the mice. Attenuated auxotrophic mutants of S. typhimurium were generated which retained their tumor-targeting capabilities. C57B6 mice with B16F10 melanomas were treated with an S. typhimurium multiple auxotroph. Tumor growth was inhibited and survival was prolonged twice that of untreated mice [9].
S. typhimurium were engineered with a lipid A mutation (msbB) and purine auxotrophic mutations (purI). These attenuated bacteria had antitumor efficacy without toxicity in mice and also had significantly reduced host TNF-a induction [10]. The S. typhimurium strain VNP20009, attenuated by msbB and purI mutations, was safely administered to patients in a Phase I trial. However, VNP20009 did not sufficiently colonize the patients’ tumors, perhaps because this strain was over-attenuated [11].
Tumor-targeting bacteria can also be engineered to express antigens for immune stimulation, express minigene DNA vaccines, express ShRNAs or other products [12–14]. Blache et al. [13] have modified S. typhimurium VNP20009 with a small hairpin RNA (shRNA) plasmid against the indoleamine 2,3-dioxygenase 1 (shIDO) which has immunosuppressive function. S. typhimurium containing shIDO silenced host IDO expression and led to tumor infiltration by polymorphonuclear neutrophils and antitumor efficacy [13].
S. typhimurium A1-R was modified to produce tumor antigen and rescued the endogenous tumor-specific CD8+ T-cell response. This increased mouse survival and rejection of established immunogenic melanomas. However, CD8+ T cells still expressed a high level of immunosuppressor PD-1. When a PD-L1 blocking antibody was administered with the tumor-antigen expressing S. typhimurium A1-R, rejection increased 80 % [14].
S. typhimurium A1-R is auxotrophic for leu-arg, which prevents it from continuously infecting normal tissues. S. typhimurium A1-R has no other apparent attenuating mutations, in contrast to VNP20009. S. typhimurium A1-R could eradicate primary and metastatic tumors as monotherapy in nude mice with prostate [15, 16], breast [17, 18], lung [19, 20], and pancreatic [21, 22] cancers, including pancreatic cancer stem cells [23] and pancreatic cancer patient-derived orthotopic xenografts (PDOX) [24], as well as sarcoma [25, 26] and glioma [27].
Treatment with tumor-targeting S. typhimurium A1-R completely prevented the appearance of bone metastasis of a high metastatic variant of breast cancer in nude mice [28–30].
2 Materials
2.1 Reagents
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1.
E. coli; JM 109.
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2.
S. typhimurium 14028 (American Type Culture Collection, Manassas, Virginia).
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3.
SOC medium.
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4.
LB medium.
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5.
EGFP gene (Clontech).
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6.
Supernatants of PT67–GFP cells, PT67–RFP (red fluorescent protein) cells and PT67 H2B–GFP cells (Clontech).
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7.
Anesthetic reagents (ketamine, xylazine, acepromazine maleate).
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8.
Kanamycin.
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9.
Nair.
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10.
pGFP (Clontech).
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11.
Restriction enzymes HindIII and NotI.
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12.
RFP cDNA (pDsRed2; Clontech).
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13.
Plasmid pLNCX2.
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14.
PT67 packaging cells (Clontech); 3T3 cells for viral titering; cell lines to be transfected with genes encoding fluorescent proteins, such as B16F0 melanoma cells (American Type Culture Collection).
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15.
Growth medium (normal and selective) appropriate for cell culture, such as DMEM.
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16.
Fetal bovine serum.
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17.
Lipofectamine PLUS (Invitrogen).
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18.
G418 neomycin.
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19.
Polysulfonic filter, 4.5 mm.
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20.
Polybrene.
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21.
Trypsin–EDTA and trypsin.
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22.
Mice expressing GFP (“GFP mice”; AntiCancer, Inc.).
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23.
Immunocompetent and immunodeficient mice (AntiCancer, Inc.).
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24.
Doxorubicin.
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25.
NaCl, 0.9 %.
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26.
Optimum cutting temperature blocks.
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27.
Antibody to rat immunoglobulin (anti-rat immunoglobulin) and anti-mouse immunoglobulin horseradish peroxidase detection kits (BD PharMingen).
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28.
Monoclonal anti-CD31 (CBL1337; Chemicon).
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29.
Monoclonal anti-nestin (rat 401; BD PharMingen).
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30.
Substrate-chromogen 3,3′-diaminobenzidine.
2.2 Equipment
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1.
OV100 Small Animal Imaging System, containing (Olympus) an MT-20 light source and DP70 CCD camera.
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2.
IV100 Laser Scanning Microscope. (Olympus)
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3.
FluoView FV1000 Confocal Microscope (Olympus).
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4.
Multiphoton tomography MPTflex™ (JenLab GmbH, Jena, Germany, and MultiPhoton Laser Technologies Inc., Irvine, CA) equipped with a tunable 80 MHz titanium–sapphire femtosecond laser (710–920 nm).
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5.
Fluorescence stereomicroscope model LZ12 (Leica).
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6.
MZ6 stereomicroscope (Leica).
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7.
C5810 three-chip cooled color CCD camera (Hamamatsu Photonics Systems).
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8.
VCR, model SLV-R1000 (Sony Corporation).
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9.
Image Pro Plus 4.0 software (Media Cybernetics).
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10.
1 ml 27G2 latex-free syringe (Becton Dickinson).
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11.
25-μl syringe (Hamilton).
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Humidified incubator with a 5 % CO2 atmosphere.
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13.
Blue LED flashlight (LDP LLC).
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14.
D470/40 excitation filter (Chroma Technology).
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15.
GG475 emission filter (Chroma Technology).
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16.
Culture dishes.
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17.
Hemocytometer.
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18.
Gene Pulser apparatus (Bio-Rad Laboratories).
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19.
Culture dishes, 60 mm; flask, 25 mm; plates, 96-well.
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20.
Cloning cylinders.
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21.
8-0 surgical suture.
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22.
D425/60 band-pass filter and 470 DCXR dichroic mirror.
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23.
Personal computer.
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24.
Coolpix camera (Nikon).
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25.
Fluorescence lightbox with fiberoptic lighting at 470 nm (Lightools Fluorescence Imaging System; Lightools Research).
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26.
Paint Shop Pro 8 (Corel) and cellR (Olympus).
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27.
BH 2-RFCA fluorescence microscope equipped with a mercury 100-W lamp power supply (Olympus) image analysis software.
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28.
CM1850 cryostat (Leica).
3 Methods
3.1 GFP Labeling of S. typhimurium (See Note 1 )
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1.
Bacteria cells (2.0 × 108) were mixed with 10 % glycerol and 2 μl of pGFP (Clontech).
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2.
Electroporation with a Gene Pulser apparatus (Bio-Rad) at 1.8 kV with the pulse controller at 1,000-W parallel resistance was used to transform S. typhimurium.
3.2 Isolation of High-Tumor Virulence Strain, S. typhimurium A1-R (See Notes 2 – 4 , 8 , and 9 )
S. typhimurium A1 auxotrophs expressing GFP were reisolated as follows:
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1.
The A1 bacteria were injected into the tail vein of a HT-29 human colon tumor-bearing nude mouse.
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2.
Three days after infection, the tumor tissue was removed from the infected mouse.
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3.
The tumor tissue was then homogenized and diluted with PBS. The resulting supernatant of the tumor tissue was cultured in LB agar plates at 37 °C overnight.
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4.
The bacteria colony with the brightest green fluorescence was isolated and cultured in 5 ml LB medium. This strain was termed S. typhimurium A1-R [17].
3.3 Adherence and Invasion Assay Comparison S. typhimurium A1 and A1-R
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1.
RFP–labeled HT-29 human colon cancer cells were grown in 24-well tissue culture plates to a density of ~104 cells per well.
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2.
A1-R bacteria were grown to late-log phase in LB broth as described previously [15].
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3.
The bacteria were diluted in cell culture medium, added to the tumor cells, and placed in an incubator at 37 °C. After 60 min, the cells were rinsed five times with 1–2 ml PBS.
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4.
Adherent bacteria were released by incubation with 0.2 ml 0.1 % Triton X-100 for 10 min. LB broth (0.8 ml) was then added, and each sample was vigorously mixed.
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5.
Adherent bacteria were quantified by plating in order to count colony-forming units (CFU) on LB agar medium.
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6.
To measure invasion of bacteria, the bacterially infected cancer cells were rinsed five times with 1–2 ml PBS and cultured in medium containing gentamicin sulfate (20 μg/ml) to kill external but not internal bacteria.
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7.
After incubation with gentamicin for 12 h, the cells were washed once with PBS, and the viable intracellular bacteria were evaluated by fluorescence microscopy [17].
3.4 Invasiveness of Dual-Color Cancer Cells by S. typhimurium A1-R (See Notes 5 and 6 )
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1.
PC-3 human prostate cancer cells labeled with RFP in the cytoplasm and GFP in the nucleus, by means of a fusion with histone H2B, were grown in 24-well tissue culture plates.
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2.
Bacteria were grown in LB and harvested at late-logarithmic phase, then diluted in cell culture medium and added to the cancer cells.
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3.
After 1 h incubation at 37 °C, the cells were rinsed and cultured in medium containing gentamycin sulfate to kill external but not internal bacteria.
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4.
Interaction between bacteria and cancer cells was observed under fluorescence microscopy magnification. The bacteria caused rapid apoptosis in the cancer cells as visualized by fragmentation of the GFP nuclei [17].
3.5 Surgical Orthotopic Implantation of Breast Tumors
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1.
Tumor fragments (1 mm3) from the MARY-X human breast tumor xenograft [31], grown s.c. in nude mice, were implanted by surgical orthotopic implantation in the mammary fat pad in nude mice. 8-0 surgical sutures are used to penetrate the tumor pieces to the fat pad.
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2.
The incision in the skin was closed with a 6-0 surgical suture in one layer. The animals were kept under isoflurane anesthesia during surgery [17].
3.6 Bacterial Targeting of Experimental Lung Metastasis (See Note 15 )
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1.
4-week-old female nude mice were used.
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2.
To obtain lung metastasis, 143B-RFP cells (1 × 106 in 100 μl PBS) were injected into the tail vein of nude mice.
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3.
On days 7, 14, and 21, 5 × 107 bacterial CFU per mouse were injected into the tail vein. Three mice were treated with bacteria, and three mice were used as untreated controls.
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4.
On day 28, all animals were sacrificed and the excised lungs were imaged using the Olympus OV100 Small Animal Imaging System (0.14× lens, excitation at 545 nm, emission at 570–625 nm) and the number of metastases were counted [26].
3.7 Orthotopic Osteosarcoma Model in Nude Mice
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1.
Four-week-old female mice were anesthetized by a ketamine mixture (10 μl ketamine HCL, 7.6 μl xylazine, 2.4 μl acepromazine maleate, and 10 μl H2O) via s.c. injection. The leg was sterilized with alcohol and an approximately 2 mm midline skin incision was made just below the knee joint to expose the tibial tuberosity [26].
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2.
5 × 105 RFP-expressing 143B-RFP cells in 5 μl Matrigel (BD Bioscience) per mouse were injected into the intramedullary cavity of the tibia with a 0.5 ml 28 G latex-free insulin syringe.
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3.
The skin was closed with a 6-0 suture.
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4.
One week after injection, a 1 cm skin incision was made over the tibia to confirm the RFP tumor growing inside the bone using the OV100 Small Animal Imaging System and the skin was closed again [26].
3.8 S. typhimurium A1-R Therapy of Experimental Pancreatic Cancer Lymph Node Metastasis (See Notes 13 and 14 )
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1.
To obtain metastasis in the axillary lymph node, XPA1-RFP cells were injected into the inguinal lymph node (afferent lymph node to the axillary) in nude mice.
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2.
Nude mice were anesthetized with the ketamine mixture via s.c. injection.
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3.
A 1-cm incision was made in the abdominal skin to expose the inguinal lymph node. The inguinal lymph node was exposed without injuring the lymphatic. The skin was fixed on a flat stand. A total of 10 μl medium containing 5 × 105 cancer cells was injected into the center of the inguinal lymph node.
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4.
Seven days later (day 0), mice were anesthetized, and the axillary lymph node was observed for metastasis. A 2-cm incision was made at the center of the chest wall. The greater pectoral muscle was released from the sternum to expose the axillary lymph node. The connective tissue on the axillary lymph node was separated. The lymph node was imaged for metastasis.
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5.
A1-R bacteria (108 CFU) were injected in the inguinal lymph node.
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6.
After injection, the axillary lymph node was observed repeatedly at different time points with the Olympus OV100 Small-Animal Imaging System. The size of the metastasis (fluorescent area [mm2]) was measured at each imaging time point [25].
3.9 S. typhimurium A1-R Therapy of Spontaneous Lymph Node Metastasis
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1.
To obtain spontaneous lymph node metastasis, 5 × 106 HT-1080-GFP-RFP human fibrosarcoma cells in 20 μl Matrigel basement membrane matrix were injected into the foot pad in nude mice.
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2.
The presence of popliteal lymph node metastasis was determined by fluorescence imaging every week after tumor injection. Mice were given the ketamine mixture for anesthesia and laid out in the prone position.
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3.
The entire limb was observed with the OV100 imaging system without any traumatic procedures. Once metastasis was confirmed in the popliteal region, bacteria therapy was started to target the metastasis (day 0).
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4.
S. typhimurium A1-R bacteria (108 CFU) were injected subcutaneously in the foot pad. The size of the metastasis and primary tumor, and body weight was measured every week. Six mice were treated with A1-R, and six were used as controls. Another two mice were used for imaging immediately and at day-7 after bacteria injection. The experiment was terminated when the control primary tumor invaded the popliteal region or the mouse died [25].
3.10 Intra-tumoral Bacterial Therapy for Pancreatic Cancer (See Notes 10 and 11 )
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1.
On day 7 after orthotopic implantation, the tumor was surgically exposed and imaged with the Olympus OV100 Small-Animal Imaging System. The size of the tumor (fluorescent area [mm2]) was measured.
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2.
Three mice were treated intramedullary (i.t). with a low concentration of S. typhimurium A1-R (107 CFU/ml), three were treated with a high concentration (108 CFU/ml), and three were used as untreated controls.
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3.
Tumor volume (mm3) was calculated with the formula V = 1/2× (length × width2).
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4.
The bacteria were injected into the tumor.
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5.
On day 14, the tumor was exposed again and the size was measured to determine the efficacy of treatment [21].
3.11 Selection of Highly Aggressive Subpopulations of XPA-1 RFP Human Pancreatic Cancer Cells (See Notes 11 and 12 )
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1.
XPA-1 RFP human pancreatic cancer cells were serially passaged in the pancreas of nude mice.
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2.
After the mice developed disseminated disease, including malignant ascites, they were sacrificed and 50 μl ascitic fluid were injected into the pancreas of another nude mouse.
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3.
After five successive passages, the ascitic fluid from the final passage was transferred into a cell culture flask containing RPMI 1640 supplemented with 10 % fetal bovine serum, 2 mM glutamine with 1 % penicillin–streptomycin. The flasks were stored at 37 °C in a 5 % incubator.
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4.
After the cancer cells were adherent, the cells were then maintained in culture as described above. This highly aggressive subpopulation of XPA-1 RFP human pancreatic cancer cells is henceforth referred to as XPA-1 RFP P5A.
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5.
Mesenteric metastases were also harvested from the same fifth generation animal and were fragmented with surgical instruments and put in a cell culture dish with RPMI 1640 supplemented with 10 % fetal bovine serum, 2 mM glutamine with 1 % penicillin–streptomycin. The flasks were placed at 37 °C in a 5 % incubator.
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6.
Once adherent cancer cells were detected via microscopy, the cells were passaged and maintained in culture as described above. This second population of highly aggressive XPA-1 RFP human pancreatic cancer cells is henceforth referred to as XPA-1 RFP P5B [22].
3.12 Intrasplenic Injection of Pancreatic Cancer Cells
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1.
Nude mice were anesthetized with the ketamine mixture injected s.c.
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2.
Human XPA-1 RFP P5 pancreatic cancer cells (5.0 × 106/50 μl Matrigel) were injected slowly as a cell suspension into the spleen of nude mice during open laparotomy. Hemostasis was then secured by gentle pressure using surgical gauze for 2 min.
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3.
The skin and peritoneum were then sutured in a single layer using 6-0 prolene sutures [22].
3.13 Dose Response of S. typhimurium A1-R Treatment of Metastatic Pancreatic Cancer
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1.
Mice with XPA1 pancreatic cancer were treated with either a low concentration of S. typhimurium A1-R (107 CFU/ml), a high concentration (108 CFU/ml), or untreated.
3.14 Orthotopic Transplantation of Red Fluorescent Protein-Expressing U87 Human Glioma Cells
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1.
For an orthotopic intramedullary spinal cord tumor (IMSCT) model, tumor fragments (0.5 mm), harvested from subcutaneously growing U87-RFP human glioma in nude mice, were implanted by surgical orthotopic implantation (SOI) into the spinal cord of nude mice.
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2.
A midline incision approximately 2 cm long was made over the midthoracic spine. Subperiosteal dissection of the paravertebral muscles was then performed.
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3.
The spinous process and bilateral lamina at the midthoracic level (T-7) were removed using a blade to expose the dura matter.
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4.
A 28-G needle was inserted into the dorsal center of the spinal cord, avoiding blood vessel injury, to create a 1 mm longitudinal incision.
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5.
The U87-RFP tumor fragment was implanted into the incision of the spinal cord.
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6.
The tumor pieces were determined to be stably expressing RFP.
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7.
The muscles, fascia, and skin were closed with a 6-0 surgical suture. After recovery, the animals were returned to their cages [27].
3.15 S. typhimurium A1-R Therapy of the Orthotopic IMSCT Model (See Notes 16 and 17 )
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1.
Five and ten days after transplantation of tumors, mice were treated with S. typhimurium A1-R (2 × 107 cfu/200 μl i.v. injection or 2 × 106 cfu/10 μl intrathecal injection).
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2.
For intrathecal injection, the mice were anesthetized and placed on a sterile field. The prominent L7 spinous process was identified through palpation of the iliac crest, and a 0.5 cm longitudinal incision was made over the dorsal lower-lumber region.
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3.
The underlying fascia was swept laterally and the spinous process at L7 and the ligamentum flavum were removed, exposing the intervertebral space.
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4.
A 33-gauge 1/2-in. removable needle connected to a 10 μl syringe (Hamilton, Reno, NV) was inserted through the dorsal L6–L7 intervertebral space.
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5.
Then S. typhimurium A1-R was injected. Eight mice were treated with S. typhimurium A1-R via i.v. injection, eight mice via intrathecal injection, and eight mice were used for an untreated control group [27].
3.16 Functional Evaluation of Hind Limbs to Determine Degree of Paralysis
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1.
Functional evaluation of hind limb strength was assessed using the Basso, Bresnahan, and Beattie (BBB) scale [32]. Mice were placed in an open field testing area and were observed for 5 min. Locomotion was rated using the BBB locomotor scale. The BBB scale ranges from 21 to 0 (21 means consistent plantar stepping and coordinated gait, consistent toe clearance, predominant paw position is parallel throughout stance, consistent trunk stability, and tail consistently up. Zero means no observable hind limb movement. All animals were tested preoperatively to ensure a baseline locomotor rating of 21.
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2.
After tumor transplantation, the animals were tested three times a week. Two different observers were randomly assigned to score the animals’ motor function. Results of the BBB score are expressed as the mean. The experiment was concluded by day-30, and all animals were sacrificed at that time. Dead animals in each group were recorded with a zero (0) functional score [27].
3.17 RFP-Expressing Murine Cutaneous Lung Cancer Model (See Note 18 )
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1.
Ten nestin-driven GFP (ND-GFP) transgenic nude mice, 6–8 weeks old, were used.
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2.
The mice were anesthetized with tribromoethanol (i.p. injection of 0.2 ml/10 g body weight of a 1.2 % solution).
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3.
RFP-expressing Lewis lung carcinoma (LLC) murine lung cancer cells (2 × 107 cells/ml) were injected into the skin of the ear, back and footpad of the ND-GFP nude mice with a 1 ml 27G1/2 latex-free syringe, 25 μl each site [20].
3.18 S. typhimurium A1-R Treatment of Tumor
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1.
Ten days after tumor growth, the tumor-bearing nestin-GFP nude mice were treated with S. typhimurium AI-R bacteria (5 × 107) via tail-vein injection. Mice without S. typhimurium A1-R injection served as untreated controls [20].
3.19 S. typhimurium A1-R Targeting of Tumor Vascularity
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1.
Lewis lung carcinoma cells (LLC-RFP) were transplanted subcutaneously in the ear, back skin or footpad of ND-GFP nude mice.
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2.
Mice were treated with S. typhimurium A1-R via tail-vein injection [20].
3.20 Experimental Lewis Lung Carcinoma Metastasis in the Lungs of C57 Mice
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1.
Female C57 immunocompetent mice, age 6 weeks, were used.
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2.
RFP-expressing Lewis lung carcinoma cells (2 × 106 in 100 μl PBS) were injected into the tail vein of C57 mice [33].
3.21 Bacterial Dosing (See Note 7 )
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1.
For treatment of Lewis lung carcinoma cells growing in the lung, either a single high-dose (5 × 107 CFU); a medium dose, 2 × 107 CFU per mouse, by weekly injection; or low metronomic dose (1 × 107 CFU) per mouse twice a week intravenously, were administered.
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2.
1 × 108 CFU bacteria were administered per mouse intrathoracically [33].
3.22 Craniotomy Open Window Model for Brain Cancer Treatment and Imaging
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1.
In order to directly treat brain metastasis or brain tumors, a craniotomy window (4 mm) was made over the right parietal bone using a skin biopsy punch. The bone fragment was removed such that the meninges and brain tissue were not injured. The craniotomy open window was covered only by the scalp where the incision was then closed with 6-0 surgical suture [34–36].
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2.
U87-RFP cells were injected into the mouse brain via the craniotomy open window to a depth of 1 mm [36].
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3.
Mice were anesthetized with the ketamine mixture (via s.c. injection.
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4.
After fixing the mice in a prone position, a 1.5 cm incision was made directly down the midline of the scalp. The scalp was retracted and the skull was exposed. Using a skin biopsy punch, a 4 mm diameter craniotomy was made over the right parietal bone.
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5.
The bone fragment was removed carefully in order not to injure the meninges and brain tissue. The craniotomy open window was covered only by the scalp. Thus, only scalp retraction was needed in order to image tumor growth in the brain.
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6.
The incision was then closed with 6-0 surgical suture.
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7.
All mice were kept in an oxygenated warmed chamber until they recovered from anesthesia [36].
3.23 Stereotactic Injection of Cancer Cells in the Brain
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1.
The mice were anesthetized with the ketamine mixture via s.c. injection.
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2.
After the craniotomy open window was made, 1 μl of a suspension containing 2 × 105 U87-RFP cells was injected stereotactically into the mouse brain using a 10 μl Hamilton syringe.
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3.
Cells were injected at the middle of the craniotomy open window to a depth of 1 mm [36].
3.24 Bacterial Therapy in the Brain Tumor Model (See Note 19 )
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1.
Two weeks after inoculation, mice were treated with S. typhimurium A1-R (2 × 107 CFU ⁄ 200 μl PBS intravenous injection from the tail vein or 1 × 106 CFU/1 μl PBS intracranial injection through the craniotomy open window) once a week for 3 weeks.
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2.
Mice were administered the same volume of PBS as the untreated control group.
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3.
After administration of S. typhimurium A1-R, fluorescence imaging was performed and changes in the diameters of the RFP-expressing tumors were recorded each week for 3 weeks.
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4.
Tumor diameters were measured each week after S. typhimurium A1-R administration.
-
5.
Tumor volume was calculated by the formula (width2 × length × 0.5). Seven mice were used in each group [36].
3.25 Mammary Fat Pad Injection of MDA-MB-435-RFP Cells
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1.
Twenty 6-week-old female nude mice, bred at AntiCancer Inc. (San Diego, CA, USA), were anesthetized with the ketamine mixture, acepromazine, and xylazine.
-
2.
MDA-MB-435-RFP cells (5 × 106/100 μl Matrigel) were slowly injected into the mammary fat pad. The needle holes were pressed in order to prevent any cancer cells overflowing and seeding at the incision site [37].
3.26 Targeting Breast Cancer Cells by S. typhimurium A1-R In Vitro
-
1.
Dual-color MDA-MB-435 cells, labeled with GFP in the nucleus and RFP in the cytoplasm, were grown on 24-well tissue culture plates to a density of 104 cells per well.
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2.
S. typhimurium A1-R were grown in LB and harvested at late-log phase, diluted in cell culture medium and added to the cancer cells [1 × 105 CFU per cell].
-
3.
After 1 h of incubation at 37 °C, the cells were rinsed and cultured in medium containing gentamycin sulfate (20 μg/ml), to kill external but not internal bacteria.
-
4.
The interaction between bacteria and cancer cells was observed at different time points by fluorescence microscopy using the Olympus FluoView FV1000 confocal microscope [37].
3.27 Antitumor Efficacy of S. typhimurium A1-R Administered by Three Different Routes (See Note 7 )
-
1.
Mice, orthotopically implanted with MDA-MB-435-RFP, were randomized into four groups. Group 1: five mice served as untreated controls; group 2: five mice were treated p.o. with 2 × 108 CFU S. typhimurium A1-R/200 μl, twice a week; group 3: five mice were treated i.v. with 2.5 × 107 CFU S. typhimurium A1-R/100 μl, twice a week; group 4: five mice were treated i.t. with 2.5 × 107 CFU S. typhimurium A1-R/50 μl, twice per week.
-
2.
The mice were sacrificed on day 34 after treatment.
-
3.
Tumor, liver and spleen were harvested and homogenized and supernatants were plated on nutrient media.
-
4.
Tissues were also prepared for standard frozen sectioning and H & E staining for histopathological analysis [37].
3.28 Reversible Skin Flap
-
1.
An arc-shaped incision (skin flap) was made in the skin to image deeper into the tumor tissue. The skin flap could be opened repeatedly to directly image the cancer cells and simply closed with a 6-0 suture [32].
-
2.
The animals were anesthetized with the ketamine-mixture [19].
3.29 Primer-Dose S. typhimurium A1-R Therapy (See Note 20 )
-
1.
Two weeks after inoculation, mice (n = 5) bearing subcutaneous tumors, were treated with S. typhimurium A1-R (1 × 106 CFU/200 μl PBS) i.v. via the tail vein as a primer dose. PBS (i.v.) was used as a control.
-
2.
Four hours after the primer dose, both control and primer dose-treated mice were treated with a high dose of S. typhimurium A1-R (1 × 107 cfu/200 μl PBS). Primer-dose, or PBS-only, followed by a high-dose was administered once a week for 4 weeks.
-
3.
After 4 weeks administration of bacterial therapy, mice were sacrificed. Tumors were removed and their size was measured [38].
3.30 Effect of S. typhimurium A1-R on TNF-α
-
1.
Tumor-bearing C57 mice were used for TNF-α determination. Blood samples were obtained at various time points after S. typhimurium A1-R dosing.
-
2.
TNF-α was measured with a mouse TNF-α enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen) [38].
3.31 Orthotopic Pancreatic Cancer Implantation
-
1.
Orthotopic human pancreatic cancer xenografts were established in nude mice by surgical orthotopic implantation (SOI) of fluorescent XPA1-RFP tumor fragments into the pancreas.
-
2.
After anesthesia, a small 6- to 10-mm transverse incision was made on the left flank of the mouse through the skin and peritoneum. The tail of the pancreas was exposed through this incision and a single 1-mm3 tumor fragment from the XPA1-RFP subcutaneous tumors was sutured to the tail of the pancreas using 8-0 nylon surgical sutures.
-
3.
Upon completion of the operation, the tail of the pancreas was returned to the abdomen, and the incision was closed in one layer using 6-0 nylon surgical sutures [23, 39–43].
3.32 S. typhimurium A1-R Therapy and Chemotherapy of Pancreatic Cancer Stem Cells (See Notes 21 – 24 )
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1.
Nude mice were orthotopically implanted with spindle-cell (stem-like) or round-cell (non-stem-like) of XPA1 as described above. The mice were treated in the following groups: (1) 5-FU (10 mg/kg, ip); (2) CDDP (5 mg/kg, ip); (3) GEM (150 mg/kg, ip); (4) A1-R (1.5 × 108 CFU/body, ip); and (5) saline (vehicle/control, ip).
-
2.
Chemotherapeutic drugs were injected weekly from day 21 after tumor implantation for 4 weeks. Each treatment arm involved eight tumor-bearing mice.
-
3.
Animals were sacrificed at 7 weeks, and tumors were weighed and harvested for analysis.
-
4.
GFP and RFP fluorescence was imaged using the OV100 variable magnification Small Animal Imaging System [44] and the FV1000 confocal microscope [19, 23].
3.33 Establishment of Patient-Derived Orthotopic Xenograph (PDOX) Model
-
1.
Pancreatic cancer tumor tissue from a single patient was originally obtained at surgery at the MD Anderson Cancer Center and cut into 3-mm3 fragments and transplanted subcutaneously in NOD/SCID mice (F1 generation) [24, 45, 46].
3.34 Establishment of fPDOX Model
-
1.
The flouresent PDOX (fPDOX) model was established, using SOI, in transgenic RFP nude mice (F2) [40, 47], from the patient tissue growing in NOD/SCID mice.
-
2.
A small 6- to 10-mm transverse incision was made on the left flank of the RFP nude mouse through the skin and peritoneum. The tail of the pancreas was exposed through this incision, and a single 1-mm3 tumor fragment from the F1 tumor was sutured to the tail of the pancreas using 8-0 nylon surgical sutures.
-
3.
Upon completion of the operation, the tail of the pancreas was returned to the abdomen and the incision was closed in one layer using 6-0 nylon surgical sutures [40–44, 48]. This model was the F-2 generation.
-
4.
The F2 tumors were harvested from transgenic nude RFP mice and passed orthotopically in non-transgenic nude mice using SOI [24].
3.35 Whole-Body Imaging Equipment (See Notes 26 – 28 )
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1.
Use an Olympus OV100 Small Animal Imaging System, containing an MT-20 light source and DP70 CCD camera, for whole-body imaging in live mice at variable magnification.
-
2.
Images are processed for contrast and brightness and analyzed with the use of Paint Shop Pro 8 (Corel Corp.) and Cell (Olympus Biosystems) [44].
3.36 Laser Scanning Microscope
-
1.
The Olympus IV100 microscope is a scanning laser microscope. A 488-nm argon laser was used. The novel stick objectives (as small as 1.3 mm) was designed specifically for this laser scanning microscope. The very narrow objectives deliver very high resolution images.
-
2.
A PC computer running FluoView software was used to control the microscope. All images were recorded and stored as proprietary multilayer 16-bit Tagged Image File Format files [20, 49].
3.37 Confocal Imaging of Cancer Cells Infected with S. typhimurium A1-R
-
1.
Confocal microscopy (Fluoview FV1000, Olympus) was used for high-resolution imaging of cancer cells infected with S. typhimurium in vitro and in vivo. Excitation sources included a cw semiconductor laser at 473 nm for GFP excitation and a tunable Mai Tai HP femtosecond laser emitting at 700–1,020 nm (Newport-Spectra Physics).
-
2.
Fluorescence images were obtained using the 20×/0.50 Uplan FLN and 40×/1.3 Oil Olympus UPLAN FLN objectives [33].
4 Notes
-
1.
GFP expression was stable for over 100 passages with GFP expression monitored at each passage [15].
-
2.
Nitrosoguanidine (NTG) mutagenesis of S. typhimurium 14028-GFP was used to obtain mutagenized bacteria that grew on complete medium, but not minimal medium.
-
3.
The type of auxotrophy was identified by growth in minimal medium supplemented with various amino acids one at a time.
-
4.
After inoculation of mice with wild-type S. typhimurium, the mice died within 2 days. The leu-arg double auxotrophic mutants allowed the inoculated mice to survive as long as control uninfected mice. Thus, S. typhimurium A1 was chosen for initial anticancer efficacy studies [15].
-
5.
Apoptosis of the cancer cells, expressing GFP in the nucleus and RFP in the cytoplasm, was readily observed by fragmentation of the GFP-expressing nuclei after infection [15].
-
6.
Two to four days after injection of S. typhimurium A1-GFP (1 × 107 CFU per mouse) in nude mice implanted with the human PC-3 prostate cancer, all observed organs were infected. By day 15, GFP-labeled bacteria could not be observed in spleen, liver, kidney, and lung. In contrast, the bacteria grew continuously in the implanted PC-3 tumor. The tumor–liver bacteria ratios were ≈2,000–10,000:1 by day 4 after injection [15].
-
7.
Three routes of administration, i.v., i.t., and p.o. of S. typhimurium A1-GFP, were compared for targeting of tumor tissue. Tumor-targeting was much higher than normal tissue and S. typhimurium A1-R-GFP disappeared in normal organs by 2 weeks using all routes [37].
-
8.
The sub-strain, S. typhimurium A1-R, was obtained [17] by passage of S. typhimurium A1 through a HT-29 and HCT-116 human colon tumors in nude mice. S. typhimurium A1-R, was found to have enhanced tumor virulence (please see below).
-
9.
The MARY-X human breast cancer was treated with S. typhimurium A1-R in an orthotopic model. Tumor regression occurred following a single i.v. injection of S. typhimurium A1-R [37]. Four of ten mice were cured [17].
-
10.
There was a significant difference between the untreated group and the high-dose-bacteria treatment group [21].
-
11.
Mice treated with S. typhimurium A1-R i.v. or intrasplenically had a much lower hepatic and splenic tumor burden compared with control mice [22].
-
12.
Locally as well as systemically administered S. typhimurium A1-R on liver metastasis of pancreatic cancer was efficacious. Mice treated with S. typhimurium A1-R given locally via intrasplenic injection or systemically via tail vein injection had a much lower hepatic and splenic tumor burden compared with control mice. Systemic treatment with intravenous S. typhimurium A1-R also increased survival time. All results were statistically significant [22].
-
13.
Just after injection, cancer cells were observed trafficking in the efferent lymph duct toward the axillary lymph node [25].
-
14.
S. typhimurium A1-R was injected into the inguinal lymph node of mice with axillary lymph node metastasis of XPA1. The bacteria targeted the axillary lymph node metastasis and all lymph node metastases were eradicated by day 7 in contrast to growing metastases in the control group [25].
-
15.
S. typhimurium A1-R was injected weekly i.v. for 3 weeks in animals with primary and lung metastatic 143B-RFP human osteosarcoma. Lung metastasis was significantly less in the S. typhimurium A1-R treatment group than control [26].
-
16.
S. typhimurium A1-R was administered to mice with the U87-RFP spinal cord glioma growing orthotopically on the dorsal side of the spinal cord where it was transplanted by surgical orthotopic implantation (SOI) [27].
-
17.
Mice were treated with S. typhimurium A1-R (by intrathecal injection). Untreated mice showed progressive paralysis beginning at day 6 after tumor transplantation and developed complete paralysis between 18 and 25 days. Mice treated with S. typhimurium A1-R i.v. had delayed onset of paralysis. With intrathecal administration of S. typhimurium A1-R had the longest delay before paralysis. The intrathecally-treated mice survived the longest, with less paralysis compared to control or i.v. treated mice [27].
-
18.
Nude mice expressing nestin-driven GFP (ND-GFP) express GFP in nascent blood vessels. The ear tumor had more blood vessels than the tumor transplanted on the back or footpad. The ear tumor was most sensitive to i.v. S. typhimurium A1-R due to increased vascularity [20].
-
19.
S. typhimurium A1-R intracranially (i.c.) inhibited brain tumor growth 7.6-fold compared with untreated mice and increased survival 73 %. Two of ten mice had their tumors eradicated [34–36]. Tumors were readily imaged by fluorescence through the cranial window [36].
-
20.
Immunocompetent mice have a different response to S. typhimurium A1-R than immunodeficient mice. Dosing of S. typhimurium A1-R had to be adjusted to avoid toxicity. In immunocompetent mice implanted with the Lewis lung carcinoma (LLC), a primer dose of S. typhimurium A1-R was first administered (1 × 106 CFU i.v.) followed by a high dose (1 × 107 CFU i.v.) 4 h later. No observable side effects were observed with the primer strategy compared to treatment with high-dose-alone. Tumor vessel destruction was enhanced by primer dosing of S. typhimurium A1-R in immunocompetent transgenic mice expressing ND-GFP, in which nascent blood vessels express GFP, along with antitumor efficacy [38].
-
21.
The XPA1 human pancreatic cancer cell line is dimorphic, containing round non-stem cells and spindle-shaped stem-like cells [23, 50]. Spindle and round cells were color-coded with fluorescent proteins of different colors.
-
22.
Spindle-shaped stem-like cancer cells were enriched in the ascites, and they were highly metastatic and drug resistant compared with the round cells. IC50 values of stem-like XPA1 cells were significantly higher than those of non-stem XPA1 cells for 5-fluorouracil (5-FU) and cisplatinum (CDDP). In contrast, S. typhimurium A1-R was active on both stem-like and non-stem XPA1 cells.
-
23.
In vivo, the combination of 5-FU and S. typhimurium A1-R significantly reduced the tumor weight of non-stem XPA1 cells. The combination of S. typhimurium A1-R with 5-FU also improved antitumor efficacy compared with 5-FU monotherapy on the stem-like cells [51].
-
24.
No significant effects on body weight, morbidity, or severe toxicities were observed in any treatment arm.
-
25.
Quiescent cancer cells are resistant to cytotoxic agents which target only proliferating cancer cells. Time-course imaging demonstrated that S. typhimurium A1-R decoyed cancer cells in monolayer culture and in tumor spheres to cycle from G0/G1 to S/G2/M, as demonstrated by fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging. After S. typhimurium A1-R decoyed quiescent cancer cells in tumor spheres to cycle from G0/G1 to S/G2/M, they became chemosensitive. S. typhimurium A1-R infection of FUCCI-expressing subcutaneous tumors growing in nude mice also decoyed the quiescent cancer cells, which were the majority of the cells in the tumors, to cycle from G0/G1 to S/G2/M, thereby making them sensitive to cytotoxic agents. The combination of S. typhimurium A1-R and cisplatinum or paclitaxel reduced tumor size compared with S. typhimurium A1-R monotherapy or cisplatinum or paclitaxel alone ([52] See Figs. 1 and 2).
-
26.
A multiphoton tomography MPTflex™ was equipped with a tunable 80 MHz titanium–sapphire femtosecond laser (710–920 nm). The optical unit consists of an active optical power attenuator to regulate the in situ power of the laser in dependence on tissue depth, an active beam stabilization device, a safety unit, and a flexible articulated mirror-arm with its compact scan head. The scan head consists of a fast galvo-scanning device to generate 2D (XY) scans, a piezo-driven z-scanner, and high NA focusing optics (NA 1.3). The optical arm is stabilized with a mechanical arm. The scan head also contains a dual-photon detector unit for the measurement of autofluorescence and second harmonic generation (SHG). The overall field-of-view of the optical system covers 350 × 350 μm2. The acquisition time for one optical section is typically 7 s. Low picojoule pulse energy is used for multiphoton excitation. The PMT1924 photodetector was used to detect signals from both fluorescence and SHG channels. LP409 and BP395/14 filter sets were used for fluorescence and SHG, respectively (filter configuration I). To separate Ds-Red fluorescence from GFP- and autofluorescence, BP593/40 and BP510/42 filter sets were used, respectively (filter configuration II) [19].
-
27.
The optics of the OV100 fluorescence imaging system have been specially developed for macroimaging as well as microimaging, with high light-gathering capacity. The instrument incorporates a unique combination of high numerical aperture and long working distance. Four individually-optimized objective lenses, parcentered and parfocal, provide a 105-fold magnification range for seamless imaging of the entire body down to the subcellular level without disturbing the animal. The OV100 has the lenses mounted on an automated turret with a high magnification range of ×1.6–×16 and a field of view ranging from 6.9 to 0.69 mm. The optics and antireflective coatings ensure optimal imaging of multiplexed fluorescent reporters in small animals. High-resolution images are captured directly on a PC (Fujitsu Siemens, Celsius W340, Model MTR-D2156). An equivalent model can also be used [44].
-
28.
Simpler systems such as a light box with appropriate filters and camera or even a blue light LED flashlight with appropriate filters can be used for macroimaging [53, 54, 55].
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Hoffman, R.M. (2015). Back to the Future: Are Tumor-Targeting Bacteria the Next-Generation Cancer Therapy?. In: Walther, W., Stein, U. (eds) Gene Therapy of Solid Cancers. Methods in Molecular Biology, vol 1317. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2727-2_14
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