* Howard Hughes Medical Institute, The Johns Hopkins School of Medicine, Baltimore, Maryland 21231; The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland 21231;
Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins School of Medicine, Baltimore, Maryland 21231;
Department of Radiology and Radiological Sciences, The Johns Hopkins School of Medicine, Baltimore, Maryland 21231; ¶ Department of Comparative Medicine, The Johns Hopkins School of Medicine, Baltimore, Maryland 21231; || Department of Oncology, University of Michigan, Ann Arbor, Michigan, 48104; ||| Cardiovascular Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
1 To whom correspondence should be addressed at the Sidney Kimmel Cancer Center, 1650 Orleans Ave. CRB, Room 590, Baltimore, MD 21231. E-mail: sbzhou{at}jhmi.edu.
Received June 28, 2005; accepted September 6, 2005
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ABSTRACT |
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INTRODUCTION |
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More recently, an attenuated strain of Salmonella typhimurium (VNP20009) has been used in a phase I clinical trial in the United States to treat metastatic melanoma and renal cell carcinoma (Toso et al., 2002). These studies failed to demonstrate significant clinical efficacy, presumably because of insufficient bacterial colonization in the tumors.
Our research group has been studying the therapeutic potential of spores derived from C. novyi-NT. C. novyi-NT is a clone derived from C. novyi after elimination of the major systemic toxin gene from the parental strain (Dang et al., 2001). C. novyi bacteria are exquisitely sensitive to oxygen (Topley, 1997
). We have shown that intravenous injection of C. novyi-NT spores into animals bearing tumors leads to hemorrhagic necrosis exclusively in the tumor tissue (Dang et al., 2001
). In immune competent mice or rabbits, the extensive destruction of tumors, coupled with an induced immune response, leads to complete eradication of tumors and cure in
30% of the animals (Agrawal et al., 2004
). In the others, the tumor regrows from a well-vascularized rim that is resistant to C. novyi-NT infection. Cures can be achieved more often in mice by combining C. novyi-NT with chemotherapeutic agents or radiation, both of which target well-oxygenated cells more effectively than hypoxic cells (Bettegowda et al., 2003
; Dang et al., 2001
, 2004
).
Oncolytic bacterial therapies can be considered purposeful attempts to convert tumors into infectious lesions. The resulting abscesses lead to the local and systemic toxicities that are expected to result from any serious infection. In our initial experiments with C. novyi-NT, we observed that 1025% of animals with large tumors died after receiving therapeutic doses of C. novyi-NT spores (Dang et al., 2001). In the present study, we describe experiments to understand the basis for the toxicity and our attempts to control it.
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MATERIALS AND METHODS |
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Animal models.
All experimental procedures were in compliance with United States laws governing animal experimentation and were approved and overseen by the Johns Hopkins University Animal Care and Use Committee. Six- to eight-week-old C57BL/6, BALB/c as well as outbred, athymic nu/nu mice were purchased from Harlan Breeders (Indianapolis, IN). Ten-week-old New Zealand White (NZW) rabbits were purchased from Myrtle's Rabbitry (Thompson Station, TN). Cage side and clinical observations of animals was performed using the "Standard Clinical Observation Criteria and Terms," which are listed in Table 1 of the Supplementary Data online. Mice were euthanized by carbon dioxide inhalation. Rabbits were euthanized by an intracardiac injection of a lethal dose of phenobarbital (100 mg/kg) after intramuscular sedation with ketamine (50 mg/kg). In some experiments, Imipenem (Merck, White House Station, NJ) was injected intraperitoneally (ip), initiated with a single loading does of 60 mg/kg followed by 40 mg/kg ip injections every 12 h through day 4 (Traub, 1988). Tissues were dissected and preserved in buffered formalin (Sigma, St. Louis, MO). Selected tissues and tumors were fixed, embedded and stained using standard histologic methods. At least five mice per treatment group were used unless otherwise indicated in the Materials and Methods or Results.
Hypoxia models.
ApoE knockout (Piedrahita et al., 1992) or ApoE/LDLR double knockout mice (Ishibashi et al., 1994
) in C57Bl/6J genetic backgrounds were obtained from the Jackson Laboratory (Bar Harbor, ME). In each model, at least two mice were evaluated after spore injection, and an equal number of mice were used as controls. These mice were fed normal chow (Harlan Teklab 18% Protein Diet), resulting in the development of aortic plaques by 18 weeks of age (Ishibashi et al., 1994
; Zhang et al., 1994
). To ensure that severe and chronic atheromatous lesions were present, mice older than 30 weeks of age were used. Myocardial infarction was induced in C57Bl/6J mice by suturing the anatomic course of the left anterior descending coronary artery one third of the length of the left ventricle from the apex, as previously described (Patten et al., 1998
). Successful interruption of coronary blood flow was indicated by immediate blanching and bulging of the affected myocardium.
Sepsis models.
BALB/c mice bearing subcutaneous CT26 tumors were injected intravenously with 100 µg Escherichia coliderived, phenol-extracted lipopolysaccharide (LPS; Sigma, St. Louis, MO) or intramuscularly with 5 x 108 Staphylococcus aureus (#25923 isolate from the Johns Hopkins Hospital Microbiology Lab). To prepare S. aureus, Mueller Hinton broth with cations was inoculated with 0.01 volumes of an overnight bacterial culture and shaken vigorously at 37°C until mid-logarithmic phase was reached. The bacteria were washed five times with normal saline before injection. Lipopolysaccharide and S. aurerus were resuspended in normal saline prior to administration. Experiments were conducted with at least three mice per group.
Blood analysis.
Blood analysis was performed in the clinical pathology laboratory at Johns Hopkins Hospital. The following blood chemistries and blood cell counts were performed on all tested mice: phosphate, uric acid, sodium, chloride, blood urea nitrogen, glucose, serum creatinine, calcium, total protein, albumin, total bilirubin, alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, CO2, white blood cell count, hematocrit, and platelet count. Potassium concentrations were not determined because the hemolysis that often occurred during blood collection made these measurements unreliable.
Tumor inoculation and spore administration.
Five million CT26 cells were injected subcutaneously into the right flank of each mouse. Tumor volume was calculated as length x width2 x 0.5. Mice were treated with C. novyi-NT spores when tumors reached 500 mm3 in size except when specifically indicated otherwise in the text. In general, 1014 days were required for tumors to reach the target size. C. novyi-NT spores were prepared as described elsewhere (Cheong et al., in preparation, Dang et al., 2001
). Rabbits were injected with 251250 x 106 spores/kg of C. novyi-NT suspended in 3 ml of normal saline (NS) via a vessel in the ear. Mice received 50025,000 x 106 spores/kg suspended in 200 µl of phosphate buffered saline (PBS) or NS via the tail vein. Experiment-specific doses are provided in the text.
Tissue processing.
Tissues from all necropsied animals were preserved in neutral buffered 10% formalin solution (Sigma, St. Louis, MO). The following tissues/organs were evaluated: adrenals, aorta, bone marrow, bone (femur), brain, cecum, cervix, colon, duodenum, epididymis, esophagus, eye, fallopian tube, gallbladder, gross lesions, harderian gland, heart, ileum, injection site, jejunum, kidneys, lacrimal gland, larynx, liver, lungs, lymph nodes (cervical, mandibular, mesenteric), mammary gland, nasal cavity, optic nerves, ovaries, pancreas, parathyroid, peripheral nerve, pharynx, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid, tongue, trachea, urinary bladder, uterus, vagina, and zymbal gland. A full set of tissues from animals in the high-dose and control groups, as well as any tissues with gross lesions in the other groups, were embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E) or with Gram-staining solution, and examined microscopically. When an abnormality in a specific tissue was observed histologically in the high-dose group, microscopic examination of the affected tissue from all the remaining dose groups was performed.
Tissue analysis.
Macroscopic and microscopic analyses were performed by a board certified veterinary pathologist (D.L.H). Liver lesions were graded as (1) background, occasional small foci of mononuclear cells; (2) minimal, less than 10% portal triads or centrilobular areas infiltrated with small numbers of mononuclear cells and occasional neutrophils; (3) mild, 1050% of portal triads or centrilobular areas infiltrated by minor populations of mononuclear cells and neutrophils; rare, randomly distributed foci in parenchyma; (4) moderate, majority (>50%) of portal triads or centrilobular areas infiltrated; limited random foci in the parenchyma comprised of a mixture of neutrophils and mononuclear infiltrates; (5) severe, extensive PMN infiltrates affecting all periportal and centrilobular areas; extensive randomly distributed foci throughout parenchyma with some coalescence of foci.
Radiolabeling studies.
C. novyi-NT spores were radiolabeled with 125I-NaI using a modification of the Iodogen method for labeling cell membrane proteins (Fraker and Speck, 1978). In brief,
3 x 109 spores suspended in 1 ml of PBS were placed in a 4-ml glass vial containing 100 µg of coated Iodogen (Pierce, Rockford, IL). Immediately after the spores were added, 5 mCi of 125I-NaI (MP Biomedicals, Costa Mesa, CA) was added to the suspension, and the contents were gently mixed. The reaction proceeded for 15 min at ambient temperature with occasional mixing. The suspension was then transferred to a 1.5-ml sterile microcentrifuge tube and briefly centrifuged at 1000 x g to pellet the spores. The supernatant was removed and the spores were washed three times with PBS. The radiochemical yield was typically 80%, and the radiochemical purity was
95%.
Fluorescent labeling of spores.
For fluorescent labeling, 50 µl of 10 mM carboxyfluorescein diacetate succinimidyl ester (Molecular Probe, Eugene, OR0 was added to 2.5 x 109 C. novyi-NT spores in a total volume of 0.5 ml. After vortexing, the suspension was incubated at room temperature for 1 h on a Labquake Rotisserie Shaker (Barnstead Thermolyne, Dubuque, IA). Unreacted fluorescein was removed by successive washes, and the spores were resuspended in PBS.
Gamma scintigraphy.
Mice were anesthetized through intraperitoneal administration of ketamine (72 mg/kg) plus acepromazine (6 mg/kg). Mice were then injected intravenously (iv) via the tail vein with 44137 µCi (1.635.07 MBq, 3 x 107 spores) of labeled spores in 200 µl of normal saline. Imaging was performed with a Gamma Medica X-SPECT small animal scanner (Northridge, CA) using a low-energy, high-resolution parallel-hole collimator. Ten-minute planar scans at 1, 3, 7, and 14 days post-injection were obtained. Prior to each scan, mice were weighed and their total radioactivity was measured with a dose calibrator (Capintec, Ramsey, NJ).
In vivo biodistribution studies.
Female BALB/c mice were injected with 2 µCi (74 Bq) of 125I-labeled spores supplemented with unlabeled spores such that the total number of spores injected was 15,000 x 106/kg. Mice in groups of three or four were sacrificed by CO2 narcosis at 1 h, 1 day, 3 days, 7 days, and 14 days post-injection, and their blood (0.1 ml), brain, heart, lungs, liver, spleen, kidneys, muscle, bone, small and large intestines, and tumor were removed, weighed, and counted in an automated gamma counter (1282 Compugamma CS, Pharmacia/LKB Nuclear, Inc, Gaithersburg, MD). The percentage of the injected dose per gram of tissue (%ID/g) was calculated by comparison with samples of a standard dilution of the initial dose. All measurements were corrected for radioactive decay.
Bacterial colony counts.
Tissues were removed, weighed, and homogenized in PBS, using 10 ml per gram of tissue and an IKA-ULTRA-TURRAX T 25 homogenizer set (IKA-WERKE, Staufen, Germany) at a speed of 24,000 rpm for 3060 s. The resulting suspension was diluted in PBS and spread onto blood agar (Brucella agar with 5% sheep blood, PML Microbiologic, Wilsonville, OR) in duplicate 50-µl aliquots. Blood was obtained from intracardiac punctures and mixed with 9 volumes of PBS, after which 50 µl of suspension was spread on blood agar plates in duplicate. Blood agar plates were incubated for 18 h at 37°C in a sealed anaerobic chamber (GasPak Systems, Becton Dickinson, Franklin Lakes, NJ). Following incubation, plates were removed from the incubator and colonies were counted. At least three mice were used for each time point. To determine whether they were derived from C. novyi-NT, any morphologically atypical colonies were evaluated by polymerase chain reaction (PCR), using the following primers to amplify the C. novyi-NT flagellin gene: 5'-AACAAATGTACAAAAAGAAATAGC-3' and 5'-CTAATCTATTTTGGATAGCTCC-3'. For determination of viability under varying oxygen concentrations,
1000 spores were spread onto blood agar plates and incubated in sealed chambers (Billups-Rothenberg, Del Mar, CA), which were purged with oxygen/nitrogen gas mixtures (Puritan Medical Products, Lithicum Heights, MD).
Statistical analysis.
The statistical significance of percent survival and toxicity was tested using the chi-squared test for trends in proportions and the Fisher's exact test. The null hypothesis was rejected if p < 0.05.
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RESULTS |
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To quantitatively assess the clearance of C. novyi-NT spores from the circulation, 125I- labeled spores were intravenously injected into healthy and tumor-bearing BALB/c mice. Radioactivity in the blood was measured at various times after injection using four mice per time point. Greater than 95% of the radioactivity was cleared from the circulation within 1 day, and activity fell to barely detectable levels over the next 2 weeks (Fig. 1A). Measurements of whole-body radioactivity showed that >70% of the injected dose was eliminated within 24 h of administration and >90% was eliminated within 14 days (Fig. 1B). No residual radioactivity was detectable 2 months after administration of spores, and there were no differences in spore clearance between healthy animals and those bearing tumors (Fig. 1A and 1B).
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To quantify this distribution, individual tissues were assessed. A significant fraction of the radioactivity was present in several tissues (particularly the lung), in addition to liver and spleen, at 1 h post-injection, but the radioactivity in these tissues disappeared quickly and was barely detectable 24 h later (Fig. 1D and 1E). Consistent with the imaging studies, the liver and spleen were the major repositories of radioactivity, and signals persisted, but gradually decreased, until the end of the experiment (day 14). The tumor-bearing mice retained radioactivity in their livers and spleens slightly longer than the healthy mice (Fig. 1D and 1E). No other differences in biodistribution patterns were observed between tumor-bearing and healthy mice in the radiolabeling studies, assessed either by imaging or by direct radioactivity measurements in excised tissues.
To define the histological distribution of C. novyi-NT spores, fluorescein-labeled spores were injected into BALB/c mice bearing CT26 tumors. After 24 h, mice were humanely euthanized, and frozen sections were prepared from brain, liver, spleen, and tumor tissues. Fluorescence microscopy showed only rare fluorescent spores in the brains or tumors of these mice, consistent with their expected presence within the vasculature. In contrast, there was a diffuse, uniform distribution of spores throughout the liver (Fig. 2, left panels). In the spleen (Fig. 2, right panels), the majority of spores were trapped within the marginal zone, corresponding to the region enriched in macrophages and other antigen-presenting cells (Aichele et al., 2003).
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Toxicological Studies in Tumor-Bearing Mice
To evaluate toxicity in tumor-bearing mice, female BALB/c mice bearing CT26 tumors were injected with 15,000 x 106 C. novyi-NT spores/kg and clinically evaluated for at least 28 days. A subset of mice that were cured of their tumors was observed for longer periods. Gross evaluation was performed at 1 h and on days 1, 3, 7, 14, 28, 90, and 365. Only hepatomegaly and splenomegaly were noted, which were maximal between days 1428 and had disappeared by 1 year (Fig. 4B). Complete histopathologic analysis of 58 organs (see Materials and Methods) at day 14 revealed only the same abnormalities observed in healthy mice that had received C. novyi-NT (25,000 x 106 spores/kg), i.e., moderate multifocal hepatitis and reactive splenic hyperplasia. All splenic abnormalities had resolved, and only background levels of hepatic inflammation remained at 365 days (Fig. 4B).
C. novyi-NT Does Not Germinate in Hypoxic Tissues That Are Not Neoplastic
The potential ability of C. novyi-NT to colonize hypoxic tissues that were not neoplastic was assessed in three different models. In the first model, 33- to 35-week-old ApoE/; LDLR/ mice were used. These mice had extensive atheromatous plaque formation in the walls of their aortas. To determine whether C. novyi-NT would germinate within these poorly vascularized plaques, mice were treated with 15,000 x 106 C. novyi-NT spores/kg and observed for 4 days, a time sufficient for extensive germination within tumor tissues (Dang et al., 2001). No signs of clinical toxicity were evident in these mice. Histopathologic evaluation of atheromatous plaques from mice necropsied on day 5 (Fig. 5) showed no evidence of necrosis or acute inflammation of the type that would be expected from bacterial colonization and that was routinely observed within tumors after treatment with C. novyi-NT (Agrawal et al., 2004
). Furthermore, Gram stains did not reveal any bacteria within or surrounding the cardiovascular lesions (Fig. 5).
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In the third model, myocardial infarcts were created in normal, 10-week-old mice by ligation of the left coronary artery. Five days later, the mice were injected iv with 15,000 x 106 C. novyi-NT spores/kg. These mice appeared clinically normal after injection. Histopathological examination of the heart 4days after injection of spores demonstrated that while the mice had the expected large myocardial infarcts with extensive areas of necrosis and fibrosis, there was no histopathological evidence of bacterial colonization within or surrounding the lesions (Fig. 5).
The Relationship Between Toxicity, Spores Dose and Tumor Size
Through the treatment of thousands of mice with C. novyi-NT spores, often in combination with other agents, it was obvious to us that toxicity, when observed, was related to the germination of the bacteria within tumors. Thus, clinical toxicity was not observed in animals without tumors, even when treated with very high doses of spores (Dang et al., 2004). Furthermore, toxicity was not observed in animals with tumors in experiments wherein bacterial germination did not occur. The toxicity most commonly manifested itself as lethargy and poor grooming, and when sufficiently severe, death within 5 days. We also noticed that toxicity appeared to be greater when larger tumors or higher doses of spores were employed.
To more quantitatively evaluate the relationship between tumor size, spore dose, and toxicity, BALB/c mice bearing subcutaneous CT26 tumors measuring between 500 and 1000 mm3 were injected with C. novyi-NT spores at 50, 500, 5000, or 15,000 x 106 spores/kg (Fig. 6A). Mortality ranged from 7.6% to 33.0% and appeared to have a dose-dependent trend that was not statistically significant (p = 0.15). In a second set of experiments, mice were segregated into groups of various tumor sizes at the time of treatment, with the smallest tumors measuring 250500 mm3 and the largest tumors measuring 15002000 mm3. Mice were injected with a fixed spore dose of 5000 x 106 spores/kg and monitored daily for toxicity (Fig. 6B). The mortality range was 030% and was greater in mice with larger tumors. This apparent trend did not achieve statistical significance (p = 0.08).
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Within 2448 h of injection, all animals that were injected with LPS or S. aureus appeared lethargic and poorly groomed, as evidenced by their ruffled fur. This was also the case, although to a lesser degree, in mice treated with C. novyi-NT (15,000 x 106 spores/kg), whereas the tumor-bearing control mice appeared healthy. All laboratory tests in the C. novyi-NT sporetreated mice, as well as in the control tumor-bearing mice, were normal. In contrast, the mice that had been injected with sublethal doses of LPS or S. aureus showed a variety of laboratory abnormalities (see Table 2 in the Supplementary Data online). Liver function tests in LPS-injected mice were elevated, with total bilirubin 25-fold higher than normal and aminotransferase (ALT) too high to be measured. In addition, mice administered LPS had low blood CO2 (<10 mEq/l), a large anion gap, a twofold increase in serum creatinine, and increases in BUN and phosphate. Mice challenged with S. aureus showed elevated ALT (49 times normal) and alkaline phosphatase levels (37 times normal).
Gross and histopathologic examinations of mice necropsied 1248 h after the injections were consistent with the laboratory abnormalities. Mice from the control, tumor-bearing group were normal. C. novyi-NT sporetreated mice had hepatosplenomegaly (as described above under Toxicological Studies in Tumor-Bearing Mice), without evidence of tissue damage, and their tissues were otherwise normal at both the gross and microscopic levels. Mice injected with LPS had hepatocellular necrosis, lymphoid necrosis in the spleen, and renal acute tubular necrosis (Fig. 7). Mice injected with S. aureus had lymphoid necrosis in the spleen but no liver or renal abnormalities. These studies indicated that the pathologic process following C. novyi-NT therapy was not that expected from typical bacterial sepsis caused by Gram-negative or Gram-positive bacteria.
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Balb/c mice bearing relatively large tumors (mean size 850 mm3) were treated with C. novyi-NT spores, and imipenem was administered 0, 6, 12, or 24 h later (Table 3). Except for the control group, mice were treated twice daily with imipenem for 4 days thereafter. The tumors in all groups of mice responded to the bacteriolytic therapy, exhibiting hemorrhagic necrosis. However, the degree of bacterial germination, as assessed by the extent of necrosis, was reduced in the animals treated with imipenem. These differential responses were reflected in tumor growth curves (Fig. 8). The tumors in animals receiving imipenem immediately after C. novyi-NT exhibited a 1-week period of delayed growth compared to animals without any spore treatment. Animals treated with antibiotics 6, 12, or 24 h after C. novyi-NT administration all had more substantial tumor responses, with tumors shrinking by an average of 6080%. Mice treated with spores alone (no antibiotics) had complete (100%) tumor regressions in all cases (Fig. 8). No toxicity was evident in the mice treated with C. novyi-NT plus antibiotics, and all mice survived. However, the tumors regrew in 19 of these 20 mice, so that only one mouse (treated with antibiotics 24 h after C. novyi-NT administration) was cured at 1 year. Control experiments showed that 20% of mice, when treated with C. novyi-NT in the absence of antibiotics, experienced severe toxicity, and 20% were cured (no tumor recurrences at 1 year). We conclude that antibiotics can indeed protect animals from the toxicity associated with C. novyi-NT infection, even when administered once robust germination has occurred, but at the expense of efficacy.
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DISCUSSION |
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Pharmacokinetics and Distribution of Spores
The pharmacokinetic profiles we observed are consistent with those predicted from previous studies of non-infectious particles delivered intravenously into animals (Proffitt et al., 1983a, 1983b
) C. novyi-NT spores are 1.3 µm long and 0.8 µm wide, in the size range that is particularly well suited for phagocytosis by reticuloendothelial cells (Proffitt et al., 1983a
). Accordingly, more than 99% of spores were cleared from the systemic circulation within 1 h. At 1 h the majority of injected spores were found in the lungs, liver and spleenthe major components of the reticuloendothelial system (RES). As expected from previous studies of particulates and other clostridial spores, clearance from the lungs was completed within a few days, and the liver and spleen remained the predominant sites of spore residence thereafter. (Lambin et al., 1998
). In tumor-bearing mice, as compared to healthy mice (Figs. 3A versus 3B), there was an increased number of viable spores cultured from the liver. The presence of a tumor may activate the RES such that it more aggressively clears and retains circulating microparticulate matter. Despite this difference in initial viable spore retention, spores in liver and spleen gradually diminished, so that none were detected 1 year later (Fig. 3).
The distribution studies also revealed that only a small percentage of spores (<1 % of the injected dose) were initially distributed within the tumor (Fig. 1E). The way in which spores, which are non-motile, migrate from the capillaries into the hypoxic regions of tumors is not clear. One hypothesis, supported by physiologic studies of tumor angiogenesis, is that the tumor endothelium is inherently leaky (Dvorak, 1990; Dvorak et al., 1988
; Hashizume et al., 2000
). Another hypothesis is that spores are ingested by phagocytic cells, which then migrate through the endothelium, carrying spores with them (O'Brien and Melville, 2000
). In either case, it is obvious that only a small number of spores are required to cause a profound tumor necrosis. In experiments wherein the dose of spores was varied (unpublished data), we estimated that as few as 10 spores within a tumor, resulting from a systemic injection of 1000 spores, could result in extensive necrosis, although the responses were not as robust, rapid, or reproducible as with the higher doses used in the present study.
Within tumors, spore counts increased 30-fold by day 1 and remained elevated as long as the tumors persisted (Fig. 3B). Note that our microbiologic assays measured only spores, not live bacteria. Because C. novyi-NT is exquisitely sensitive to oxygen (see Figure 1 in the Supplementary Data online), we found it difficult to homogenize tissues in a fashion that preserved the hypoxic environment of tumors and would provide reliable recovery of live bacteria. The increase in spore number within tumors reflected the fact that germination within tumors is followed by sporulation, as confirmed by microscopic analysis of tumors. The release of live bacteria and spores from the tumors is the likely basis, in part, for the increased number of viable spores in the liver (Fig. 3) and the significant hepatosplenomegaly observed in tumor-bearing animals after injection of spores (Fig. 4B). An important finding of the present study was that spores were eventually cleared from all organs, even in tumor-bearing animals.
Minimal Toxicity in Animals Without Tumors
Our first step in investigating the potential toxicity of C. novyi-NT spores involved traditional acute toxicology studies in two animal species. Despite the high doses of spores used in these experiments, no clinical evidence of toxicity was observed in any animal. Gross pathologic examination was also unremarkable, except for mild hepatosplenomagaly in the high-dose groups. Microscopic examination showed hepatic and adrenal inflammatory changes at day 14 that were already resolving by day 28 and had disappeared by 1 year.
The second step in toxicological investigation involved the analysis of mice with hypoxic lesions that were unrelated to neoplasia. Three different mouse models were tested, two incorporating atheromatous lesions and one incorporating myocardial infarction. Except for the expected atheromas and infarctions, no clinical or pathological abnormalities were detected in mice inoculated with C. novyi-NT spores in any of the animals. Moreover, no microscopic signs of bacterial germination or growth were observed within or surrounding these lesions. It is of interest in this regard that parental C. novyi spores are able to infect penetrating wounds induced by gunshots or contaminated drug injections (Boyd et al., 1972a, 1972b
; Majumdar et al., 2004
; McGuigan and Roworth, 2002
; McGuigan et al., 2002
; Mulleague et al., 2001
). Tumors have been likened to "wounds that will not heal" (Brown et al., 1999
; Metheny-Barlow and Li, 2003
), and it is possible that the microenvironment within tumors is more like that observed in fresh wounds than that in fresh infarcts or in poorly vascularized tissues like atheromas. The mechanism(s) underlying the ability of C. novyi-NT spores to germinate within the hypoxic regions of tumors (or wounds) but not within other hypoxic regions is not known, but several can be imagined. First, it is possible that the oxygen levels, though lower than normal, are higher within the atheromatous/infarcted tissues than in tumors; as noted above, C. novyi-NT is exquisitely sensitive to oxygen (see Figure 1 of the Supplementary Data online). Second, it is possible that C. novyi-NT spores cannot enter into the hypoxic regions within atheromatous or infarcted tissues because the vasculature is not leaky, as it is in tumors. And third, it is possible that some other feature of tumors or wounds, unrelated to oxygen levels or circulation per se, allows C. novyi-NT germination. In experiments in vitro, we have shown that C. novyi-NT can germinate when co-cultured with cancer cell lines in ambient air, but not when co-cultured with normal cell lines (unpublished data). The factors responsible for this tumor cellspecific germination are not understood.
Toxicity in Animals with Tumors
In contrast to healthy mice or those with atherosclerosis or myocardial infarction, mice bearing tumors did mount a pathologic response to treatment with C. novyi-NT. All available evidence suggests that the response was mediated by germinated C. novyi-NT bacteria, not the spores. Clinically, lethargy and poor grooming were commonly seen, and hepatosplenomegaly was observed pathologically. The inflammatory changes observed in the liver and spleen were those expected from infection with a relatively indolent pathogen and were fully reversible with time in surviving animals (Figs. 4A and 4B).
It is not surprising that large numbers of germinated C. novyi-NT bacteria within tumors cause toxicity in animals. Such toxicity has been established in many examples of infectious diseases (Lolis and Bucala, 2003; Ward, 2004
), and its pathogenesis focuses on the cytokine storm induced in the host. The intensity of the inflammatory response leads to the release of a variety of vasoactive and immunomodulatory factors that cause sepsis or systemic inflammatory response syndrome (SIRS) (Mitaka, 2005
; Rice and Bernard, 2005
). Previous studies have demonstrated the release of inflammatory cytokines during therapy with C. novyi-NT (Agrawal et al., 2004
). Based on studies of various infections, it would be expected that larger amounts of bacteria would result in higher levels of cytokines, and this is consistent with our observation of a relationship between the level of toxicity and both tumor size and the dose of the spore inoculum (Fig. 6). It is notable in this regard that the mouse tumors used in our experiments were much larger relative to body weight than would occur in larger animals. Perhaps this explains why treatment-related mortality from C. novyi-NT spores was not detected in rabbits (Agrawal et al., 2004
). In addition, even in mice with extremely large tumors (greater than 10% of body weight), mortality rates plateaued at 30%. This may represent the self-limiting nature of an infection that destroys the anaerobic environment on which the bacteria depend for survival. The lack of the pathogenic
-toxin also limits the absolute toxicity of C. novyi-NT (Dang et al., 2001
). Viewed in the context of other infectious diseases, it is therefore remarkable that the very large abscesses caused by C. novyi-NT in tumor-bearing mice were tolerated by most of the animals.
Minimizing Toxicity in Tumor-Bearing Mice
To begin to understand the basis for the toxicity observed after treatment with C. novyi-NT, we compared clinical, pathologic, and laboratory data obtained in animals treated with C. novyi-NT to the data obtained in mice administered agents known to cause typical SIRS. All mice injected with either LPS or S. aureus appeared ill and displayed poor grooming and lethargy. To a lesser and more variable extent, the mice treated with C. novyi-NT also displayed these symptoms. As expected, mice injected with LPS or S. aureus showed pathologic hepatic and renal abnormalities consistent with SIRS and laboratory findings suggestive of hepatic and renal injury. Surprisingly, clinically ill tumor-bearing mice treated with C. novyi-NT showed no such findings.
Toxicity from bacterial infections can generally be reduced by treating the infections with antibiotics. The toxicity from C. novyi-NTinduced toxicity was no exception. Antibiotics did indeed minimize toxicity, and no deaths were observed in animals treated with antibiotics, even when antibiotics were not initiated until after a large number of bacteria were present within the tumor (24 h after C. novyi-NT administration; Fig. 8). But antibiotics also reduced the efficacy of the treatment by limiting the number of tumor cells killed by the bacteria. All tumors in the antibiotic-treated mice showed evidence of central germination and necrosis, but a larger rim of viable tumor remained. We propose that antibiotics cannot penetrate the hypoxic avascular core of the tumor but do penetrate the vascular tumor rim and suppress bacterial growth, limit inflammation, and reduce complete tumor debulking.
Fluid resuscitation is another cornerstone of the therapy of severe infections (Dellinger et al., 2004; Remick et al., 2005
; The SAFE Study Investigators, 2004
). Fever, anorexia, and an increased metabolic demand place stress on animals during infection and can result in fluid loss. The cytokine storm (Agrawal et al., 2004
; Remick et al., 2005
; Schrier and Wang, 2004
), together with this fluid loss, makes maintaining circulatory volume and blood pressure essential for survival. Based on the absence of distinct laboratory and histopathologic abnormalities in mice treated with C. novyi-NT spores, we suspected that hypovolemia with hypotension from fluid loss and cytokine-mediated vasodilatation was largely responsible for the deaths that were observed. This idea was consistent with clinical observations: lethargy and general malaise could have resulted in the animals' drinking insufficient quantities of water, and ruffled fur can be a sign of dehydration. This hypothesis was also consistent with the fact that the toxicity was less evident in rabbits, which are larger animals with lower metabolic rates and are thereby more tolerant to dehydration.
In the present study, we showed that fluid resuscitation alone could effectively rescue animals from death. As the infection within the tumors is self-limiting, it was only necessary to administer fluids for a few days. Our finding that the toxicity in tumor-bearing mice is reversible will be useful for future studies employing C. novyi-NT in combination with other therapeutic agents. It is likely that this simple measure would also reduce the toxicity of other forms of therapy based on relatively nontoxic bacteria or components derived from them. Most importantly, the lessons learned in mice should be applicable to humans if and when Clostridia-based therapies re-emerge in the clinical setting.
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