COBALT: Combination Therapy Bacteriolytic = chemo and successful in animal bacteria but also criticized by high toxicity

This post transferred from cancer news, though placed there in 2001, to current mainstream research here by September 7, 2004. We have all possible information from PubMed removed and added, including the discussion and treatment protocols used, etc. This approach is a combination of bacteria and chemo oi in line with eg the Newcastle virus , already in phase II trials and proven effective the adenovirus , where British researchers recently experimenting. Under the old post much new information. Yahoo also a critical consideration of a fellow researcher, who oi a few sensitive points of this method As indicated. Too bad that apparently after 2001 it went no further, at least we can find no further publications on this matter. The following was published: November 27, 2001, 10.1073/pnas.251543698
Proc Natl Acad Sci U.S. A. 2001 in December 1918, 98 (26): 15155-15160 DOI: 10.1073/pnas.251543698

It has already been reported this news on the Internet, etc., but now is also in the Medical Journal reported this. A major breakthrough seems to be achieved in treating cancer using a combination of chemotherapy and a certain type of bacteria within 24 hours (!!!!) in 7 of 8 mice was dying of cancer tumors completely. Now has been known that cancer cells can not stand oxygen. It is no coincidence that people with cancer are advised to walk a lot, play sports and in some privèklinieken, eg in Germany, oxygen in the blood artificially placed to support the treatment. Also Professor Warburg, Nobel laureate, showed that cancer cells can not grow in an oxygenated environment. This little message seems to be one of many, but in my opinion this is a very important discovery, and especially since mainstream scientists finally see the usefulness. Here is the article in the journal Medicine and below the English abstract and description of the university where this study took place. Italics are additions to the letter sent me this person, thank you, of course.

Source Journal of Medicine:

Anaerobic bacteria destroy cancer cells. (Anaerobic means that they have a dislike of oxygen - they die in the presence of oxygen)

Chemotherapeutic agents are hardly capable of neoplastic cells (cancer cells so) in poorly vascularized (perfused) areas of tumors to kill.
Dang et al have 26 different types of anaerobic bacteria tested for their ability to anoxic (non oxygenated) areas of tumors to grow and destroy surrounding neoplastic cells. Clostridium Novyi-NT (C. Novyi a variant that produces no toxin) proved very good at this. In combination with conventional chemotherapeutic agents was usually within 24 hours of extensive haemorrhagic (accompanied, due to, characterized by bleeding) necrosis (tissue - dying), resulting in a prolonged anti-tumor effect of treatment. Some tumors were already in complete regression after treatment. This method, the researchers' combination bacteriolytic therapy (COBALT) call may be an entirely new way to fight cancer. (Proc Natl Acad Sci USA 2001, 98:15155-60)

Source: Website of John Hopkins University

Zeroing In on Tumors With "Smart Bombs" or Bacteria
Scientists at Johns Hopkins's Sidney Kimmel Comprehensive Cancer Center have favored recruiting an Unlikely ally in the war against cancer: a genetically modified soil bacterium, Clostridium Novyi (C. Novyi). Clinicians one day May Be Able to use it as a kind of biological smart bomb against tumors.

Hopkins Oncology professors Bert Vogelstein and Kenneth Kinzler are Developing the new approach, Which They Call COBALT (for combination bacteriolytic therapy), to take advantage of the oxygen-poor environment inside some cancers.

Some advanced cancers grow so Quickly That They Can not coerce the body write enough new blood vessels growing to support the tumor cells. This can-leave areas inside the tumors with poor blood circulation, low levels of oxygen, and masses of dead and dying cells.

Chemotherapy drugs have a hard time overreaching Such areas Because of the poor blood circulation, and radiation therapy relies on the presence of oxygen to trigger cell death. Axis a result, after traditional treatments stop, can-cancerous cells start growing again.

Vogelstein, who is the Clayton Professor of Oncology and a Howard Hughes Medical Institute investigator, said the idea of Using bacteria to combat cancer Germantown Originated with scientists, who unsuccessfully tried to attack tumors with bacteria about 50 years ago. Vogelstein and Other Hopkins researchers, Renewing the Attempt to Develop this approach, about a year ago Began systematically screening bacteria distance or oxygen-poor environments, searching for a species to thrive That Would Such environments and kill tumor cells.

They settled on C. Novyi as the best candidate but found partially-They first had to defang the virus by genetically Removing ITS ability to produce a toxin with Potentially lethal side effects. Tests in mice with tumors showed the bacteria Almost Worked Exactly as they'd Hoped, wiping out cancerous tissue within-cells, but dying out as it approached the oxygen-rich healthy tissue on the perimeters of the tumor.

"The idea is to selectively attack the thesis tumors with bacteria from inside and from the outside with chemotherapy," said Vogelstein.

The combined approach Produced dramatic results in mice, Destroy More Than half the tumors Treated Within 24 Hours. In a paper published in the Proceedings of the National Academy of Sciences on November 27, 2001, the researchers Reported That COBALT treatments shrank or eliminated tumors in seven or eight mice. One mouse relapsed but responded well to a second treatment.

Co-author cautioned Kinzler Several Years of Research That Will Be Necessary before scientists can-start human clinical trials or Cobalt.

"We Hope that this compromise research add a new dimension to cancer treatment but Realize That the way tumors respond to treatment in mice can-Than be different in humans," Kinzler said.

Researchers must learn Which Also chemotherapy agents work best compromise with the bacteria, and can-whethere They Use other drugs to combat the effects of toxins created by the rapid destruction of tumors, a phenomenon Known As tumor lysis. - Michael Purdy

Doctors and oncologists details of this experimental approach will can directly contact The Johns Hopkins Oncology Center, 1650 Orleans Street, Room 589, Baltimore, MD 21231-1001. E-mail: vogelbe@welch.jhu.edu .

Here is the abstract of the study described and published:

Medical Sciences
Combination bacteriolytic therapy for the treatment of experimental tumors
H. Long Dang, Chetan Bettegowda, David L. Huso, Kenneth W. Kinzler, and Bert Vogelstein *
The Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Division of Comparative Medicine, The Johns Hopkins School of Medicine, and The Johns Hopkins Oncology Center, 1650 Orleans Street, Baltimore, MD 21231

Contributed by Bert Vogelstein, October 12, 2001

Current therapeutic approaches for cancer chemotherapy are limited in part by the inability of drugs to destroy cells within-Neoplastic Compartments or poorly vascularized tumors. We have here systematically assessed anaerobic bacteria for Their capacity to grow expansively within-avascular Compartments or transplanted tumors. Differentiate among 26 strains tested, one (Clostridium Novyi) appeared Particularly promising. We created a strain of C. Novyi ITS devoid of lethal toxin (C. Novyi-NT) and showed That intravenously injected C. Novyi-NT spores germinated within-the avascular regions of tumors in mice and surrounding 'Destroyed viable tumor cells. When C. Novyi-NT spores Were Administered together with Conventional chemo therapeutic drugs, extensification or hemorrhagic necrosisOften tumors Developed Within 24 h, tion results in significant and prolonged antitumor effects. This strategy, called combination bacteriolytic therapy (COBALT), HAS the potential to add a new dimension to the treatment of cancer.

Introduction

Despite Enormous progress in understanding the pathophysiology of neoplasia, advanced forms of cancer in pubs recalcitrant to treatment. Although the basis for this failure is complex, One Reason Is That must containerization large tumors, poorly vascularized areas That limit the efficacy of radiation and chemo therapeutic drugs (1). The poorly vascularized regions are less sensitive to ionizing radiation becauses ITS cell-killing effects depend on oxygen; They are less sensitive to chemotherapeutic drugs becauses therapeutic drug delivery to regions thesis is suboptimal. Because a cancer therapeutic agent must not leave significant clusters of viable cells within-Any lesion to Achieve a Clinically meaningful effect, the poorly vascularized regions of tumors represent a Major Obstacle to effective treatment.

One of the Most Important Recent Developments in tumor biology is the recognition That neoangiogenesis is essential for the growth of tumors to Clinically meaningful sizes (2). What is less well recognized Is That neoangiogenesis Often this does not keep pace with the growth of the Neoplastic cells, tion result in large necrotic areas composed of dead or dying cells. For example, we found That EACH or 20 Randomly selected liver metastases> 1 cm 3 in size contained Relatively large avascular regions in general constituting 25-75% of the tumor mass (Fig. 1). Importantly, cells adjacent to necrotic areas are poorly vascularized thesis and Likely To Be Difficult to treat with Conventional agents.
, Br> In the work described here we exploit the fact That Attempted to necrotic regions exist only within-not in normal tissues and tumors. We wished to initially develop a toxic agent That Could Be thesis specifically delivered to areas and, in theory, could-kill surrounding 'viable tumor cells. We chose to Investigate anaerobic bacteria for this purpose. It Has Been Recognized for half a century That Could Such bacteria proliferate selectively in the hypoxic regions of tumors (3-18). Clever strategies for Exploiting Potentially Such bacteria for diagnostic and therapeutic Purposes s been devised, although Relatively little work in this area HAS Recently tasks place. We Hoped That a systematic screen for Appropriate anaerobic bacteria That could-kill tumor cells adjacent to the poorly vascularized regions, Rather Than Just Such localize to regions, Would rejuvenate interest in this approach. Further More, We Hoped That chemo therapeutic agents That killed the well vascularized regions of tumors, When Administered in Conjunction with 'appropriate bacteria, Would result in the destruction of a major or proportioning Neoplastic cells within-the tumors. Our Progress Toward Realizing thesis goals is described below.

Materials and Methods

Bacterial Strain and Growth. The bacterial strains in this study Were Tested Purchased from the American Type Culture Collection and are listed in Table 1. All bacteria Lactobacilli Forman Were grown anaerobically in liquid cultures at 37 ° C in Reinforced Clostridial Medium (RCM) (Difco). Were lactobacilli grown in Lactobacilli MRS broth (Difco). Intravenous injections of bacteria gene rally included 5 x 107 bacteria suspended in 0.5 ml Dulbecco's PBS (Life Technologies). Mice That Received injections or Bifidobacteria Were Also Given ip injections of lactulose daily for 5 days to increasefontsize bacterial growth (16). Intratumoral injections of bacteria gene rally included 1 x 107 bacteria suspended in 0.1 ml of PBS.

Drugs. Dolastatin-10 (D10) was provided by George R. Pettit (Cancer Research Institute, Arizona State University, Tempe, AZ), Gregory P. Kalemkerian (Department of Internal Medicine, Wayne State University, Detroit), and Robert J. Schultz (Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda). Combretastatin A-4 was kindly provided by Robert J. Schultz. Cytoxan (CTX), mitomycin C (MMC), vincristine, colchicine, and vinblastine chemo therapeutic agents are Commercially available (Sigma).
, Br> Cell Lines and Animals. Female athymic nude mice and C57Bl6 6-8 weeks of age Were Purchased from Harlan (Indianapolis). HCT116 colon cancer cells and B16 melanoma cells Were grown as monolayers in McCoy 5A medium (Life Technologies, Rockville, MD) supplemented with 5% FBS and 1% penicillin / streptomycin (catalog No. 15140-122).

Sporulation and Generation of a Nontoxigenic C. Novyi Strain. Spores of Clostridium Novyi generated by growing the strains Were Organisms anaerobically at 37 ° C, pH 7.4 in a medium containing 5 g of Na2HPO4, 30 g peptone, 0.5 g L-cysteine, 10 g maltose, and 5% wt / vol dried cooked meat particles (Difco) per 1 liter. After1 week in this medium, spores settled in the cooked meat particle layer (19). Spores Were Further purified from contaminations tion vegetative forms on a discontinuous Percoll gradient. To remove the lethal toxin gene from the wild-type C. Novyi strain, C. Were Novyi spores heated at 70 ° C for 15 min to inactivate the phage Carrying the toxin (20, 21). Were the spores then plated on reinforced clostridial medium agar and incubated anaerobically at 37 ° C for 48 h. Were Isolated colonies cultured in liquid RCM for another 24 to 48 h and then Tested for the presence of the lethal toxin gene by PCR.

In Vivo Studies. Six to eight week old female BALB / c athymic nude mice or C57Bl6 Were implanted with sc tumors through the injection of 2.5 x 106 HCT116 or B16 cells, respectively. After 8-12 days of tumor establishment, treatment was initiated with spores or drugs. Screening of bacterial strains for Their ability to populate tumor grafts was done by Either intratumoral injection (100 μl volume, 1 × 107 bacteria) or iv injection (500 μl volume, 5 × 107 spores or bacteria) enter the tail vein. C. Novyi-NT spores and D10 Were diluted to the 'appropriate concentration in PBS and then Administered by iv injection in a volume of 500 aliquot. CTX and MMC in PBS and then diluted Were Given by ip injection in a volume of 500 aliquot. Tumor growth was assessed by measuring the size of the major and minor axes of sc tumors everytime everytime 4 and 2 days for B16 and HCT116 tumors, respectively, Using calipers. Tumor volume was then Calculated by Using the equation length × 0.5 × width2.> Br>
Results

Choice Of Bacterial Species. From previous studies it was clear That species of anaerobic bacteria grow could-within-the-hypoxic regions of tumors. An example is provided by Bifidobacterium longum, Which, When injected intravenously Writing mice with sc tumors, overgrew specifically and robustly within-within-the tumors but not normal tissue (16, 17). Gram stains of sections of the tumors, however, revealed bacteria must That Were Tightly clustered within-Rather Than colonies distributed throughout the necrotic regions (Fig. 2 A and B). Considered as we dispersion of the bacteria essential to Achieve the Desired effects, numerous anaerobic species of three genera differential Were Tested in an effort to find one (s) Exhibiting this phenotype (Table 1). For this purpose, Bifidobacterium and Lactobacillus strains Were injected intravenously, whereas Clostridium strains, Which are Generally highly toxic When injected intravenously, injected directly Were Writing tumors. Among the 26 strains listed in Table 1, only two (C. Novyi and C. sordellii) exhibited extensification spreading throughout the poorly vascularized portions of the tumors (data not Shown). Although this spread was undoubtedly facilitated by the motile nature of theses two species, other motile anaerobic bacteria, include Other Clostridium strains, did not exhibit this property When Tested under Identical conditions.

Infiltration of the Tumor Mass Following iv Injection of C. Novyi Spores. For an experimental therapy to represent a Potentially viable tool for the treatment of disseminated cancers, it must have the capacity to be delivered systemically Rather Than through local, intratumoral injection. Although live bacteria are toxic Often When injected intravenously, it Has Been Shown That bacterial spores are nontoxic to normal animals. Accordingly, we found That large numbers (up to 108 in a volume of 500 microl) or C. Novyi and C. sordellii spores Could Be Normal mice injected intravenously Writing Without Causing Any notice-able side effects. When intravenously injected mice with sc B16 tumors Writing, however, C. Novyi bacteria floridly germinated within-within-the tumors 16 h (Fig. 2C). In contrast, no germinated bacteria Were observed in the liver, spleen, kidney, lung, or brain or thesis mice (data not Shown). Similar results Were observed after iv injection of C. sordellii spores (data not Shown).

Genetic Modification or C. Novyi. Although C. Novyi and C. sordellii spores Both had the capacity to grow within-tumors and kill some surrounding 'tumor cells, there was at least one "small" Encountered problem with this experimental treatment: 16-18 h following the initiation of treatment, all of the mice died. We Suspected thats the cause of death was the release of potent lethal toxins from the bacteria germinating within-the tumors. Indeed, other anaerobic bacterial spores have Proved highly toxic to animals and humans following germination within-the-anaerobic environments present in tumors or wounds, and the resultant mortality was Shown to be due to specific secreted toxins (22-26).

To mitigate systemic toxicity, we Attempted to Eliminate the lethal toxin gene from C. Novyi. We chose C. Novyi Rather Than C. sordellii for this purpose Because The Latter HAS two homologoustoxin genes (27) Rather Than Because one and the single C. Novyi toxin gene is located within-a phage episome (20, 21, 28). Bacteria Were heat treated to induce loss of the phage agar plates and inoculated failure. Of 400 bacterial colonies screened, three Were observed to have lost the toxin gene When assessed by Using PCR toxin gene-specific primers (examples in Fig. 3). Phospholipase C, a C. Novyi gene contained within-the-bacterial Rather Than the phage genome (29), served as control for this PCR experiment. One clone, named C. Novyi-NT, That had lost the toxin gene was selected FE analysis.

Destruction of Tumor Cells Following Injection of C. Novyi-NT Spores. C. Novyi-NT spores devoid of the lethal toxin injected intravenously Were Writing mice with tumors. These spores retained Their capacity to germinate within-tumors and resulted in greatly expanded areas of necrosis (Fig. 4 A vs.. B). However, spores thesis, unlike Those of Their Parents, nontoxic Were When injected alone, with no ill effects observed after generation rallied injection or up to 108 spores Writing mice with tumors. In contrast, all mice died after injection of 5 x 107 parental C. Novyi spores Writing mice with tumors. Growing bacteria could-be observed throughout the much-enlarged necrotic regions of tumors after injection of spores (data not Shown). The Enlargement of the necrotic regions was apparently due to the destruction of viable tumor cells adjacent to the original necrotic regions by the bacteria. Indeed, a bacterial "film" (30) was routinely observed at the Interface Between the necrotic area and the remains of the viable tumor rim, as if the bacteria Were Destroy the viable tumor cells and Using ITS degradation products as nutrients (Data Not Shown ). This tumor infiltration effect was similar To that observed with wild-type C. Novyi bacteria (Fig. 2D).

Combination Therapy. Ash noted in the introduction, we Hoped to combine a bacterial agent with more Conventional chemo therapeutic agents in an effort to attack the tumors from Both the inside and outside, respectively. Following preliminary investigation with Several Such agents, we concentrate rated on two classes: (i) DNA Damaging Agents, Such as MMC and CTX, That selectively kill tumor cells, and (ii) Agents That Appear to partially-collapse tumor vasculature, Such As Flavone acetic acid derivatives and microtubule binding agents (31, 32). The class of agents Latter Has Been Shown To Be Able to clean interfering with circulation through the tumors and thereby trap large molecules, Such as antibodies or bacteria, That Have gained access to the tumor tissue (33-35). Among Flavone acetic acid and the microtubule-binding agents Tested (include vinblastine, vincristine, colchicine, combretastatin A-4, and D10), D10 appeared to have pronounced effects and the wine was Chosen for Further experimentation.

Xenografts of the colorectal cancer cell line HCT116 Were Used To test the effects of this combination therapy in nude mice Because The tumors Could Easily be visualized under the hairless skin. Shaft Shown in Fig. 5, sequential treatment with C. Novyi-NT spores, D10, and MMC resulted in dramatic effects on large sc tumors, Easily observable through the skin. Twenty four hours after the injection or C. Novyi-NT spores, the tumor mass swelled and Became edematous (Fig. 5A). Six hours after receiving D10, a black spot Developed near the center of the tumor, represents an area of hemorrhagic necrosis. This spot expanded in size and within-24 h Often Completely enveloped the tumor (Fig. 5A, day 1). Hematoxylin / eosin (H & E) staining of sections of tumors revealed thesis extensification destruction of the tumors, or at accompanied by infiltration of inflammatory cells (data not Shown). These necrotic masses then shrank over a period of 2-4 weeks (Fig. 5A, 14-30 days). In many mice, synthesis necrotic masses and Disappeared Eventually dissolved, leaving the animals tumor-free (Fig. 5B Quantified below). Similar, although less dramatic, results Were observed following the sequential treatment with C. Novyi-NT and D10 (without MMC), but never with D10 and MMC in the absence or C. Novyi-NT and with C. Rarely Novyi-NT alone.

The dramatic antineoplastic effects of this combination bacteriolytic therapy (COBALT) Were associated with significant toxicity. Approximately 15% of animals with tumors of 350 mm3 in size died within-COBALT 24-72 h or receiving. Clearly this toxicity was related to the size of the tumors, becauses ≈ 45% of animals with larger tumors (≈ 700 mm 3) died. Deaths Were not observed after administration of C. Novyi-NT spores alone or with chemotherapy alone. Although the reason for the deaths of animals was not clear thesis, They May Have Been due to tumor lysis syndrome, a phenomenon observed in the clinic Previously When large tumor burdens are Rapidly Destroyed By antineoplastic agents (see Discussion).

Theantineoplastic effects of COBALT Quantified Were Shown in the experiments in Fig. 6. Animals with Relatively large sc HCT116 tumors (starting tumor volume ≈ 700 mm 3) Were Treated with drugs alone (D10 plus MMC) or C. Novyi-NT spores plus the drugs. Ash Can Be seen in Fig. 6A, the drug alone Slowed the growth of the tumors, although the tumors Continued to grow and the animals had to be killed at 10-14 days, When tumor weights Exceeded 10% of body weight. The Addition or C. Novyi-NT spores Dramatically enhanced the effects of treatment, with tumors shrinking Actually Rather Than Simply slowing. In the Experiment Shown in Fig. 6A, seven or eight animals had dramatic tumor regressions after only one administration of COBALT, and four of the five animals survived the therapy That Were Completely cured, with no evidence of tumor regrowth after a Further three months time. Also significant tumor shrinkage was seen When mice Were Given sequential treatment with C. Novyi-NT spores plus D10 (Fig. 6B). However, there was no long-term tumor-free survival and the treatment had to be repeated once everytime the full 2 weeks unles combination, include MMC, was included. This repetition was associated with additional toxicity, include deaths or ≈ 15% of animals with Each dose. The full COBALT regimen was therefore preferred on the basis of overall Reduced Increased efficacy and toxicity.

To determining whethere COBALT Would Affect Other tumor types, we Treated C57Bl6 mice with large syngeneic B16 tumors. In this case, CTX was substituted for MMC, becauses Were B16 tumor cells more sensitive to CTX Than to MMC. The drugs alone had some antitumor effects, as expected, although the tumor Continued to grow in size and the animals had to be killed Within 1 week after beginning therapy (Fig. 6C). C. Novyi-NT spores considerably enhanced effects hypothesis: Were the tumors observed to shrink Rather Than Simply enlarge at a Slower Rate (Fig. 6C). D10 plus C. Novyi-NT spores (without CTX) had significant antineoplastic effects on B16 tumors, but the Addition of the tumor cytotoxic agent (CTX) Further enhanced the efficacy of COBALT (Fig. 6C). In the B16 tumor model, maintenance COBALT (once weekly) was required to keep from the tumors to regrow, whereas HCT116 cells with a single treatment cured about half the mice.

Discussion:

The results recorded above show can-COBALT That resulted in rapid and dramatic regressions of experimental tumors in mice. Just Relatively large tumors Could Be Treated successfully with COBALT, although tumors of the size Used in our experiments do not respond well to rally beyond chemo therapeutic agents (Figs. 5 and 6). Also it is clear many questions That Remain. For example, the basis for the potent tumor cell killing in the Vicinity of the germinating bacteria is not Understood. That we found many Other bacterial strains germinate could-within-the-necrotic regions of tumors but did not exhibit this potent cytotoxic activity. Clearly this killing is not due to the lethal toxin gene or C. Novyi, Because this gene was deleted in the C. Novyi-NT strain Used in COBALT. IT would be interesting in the future to qualification Which of the C. Novyi-NT genes are Responsible for thesis tumor cytolytic effects. Another point of interest That was an agent acting on the vasculature (D10) considerably enhanced the ability of C. Novyi-NT spores to lyse tumors. Presumably, the vascular collapse Further lowered the oxygen tension near the trapped bacteria and thereby Increased the potential for bacterial growth. Indeed it Has Been That demonstrated vascular collapsing agents can-increasefontsize the germination of bacterial spores in tumors (33). D10 was Given the bacterial spores after Rather Than before Because We Believed That partial vascular collapse before spore administration Might have a deleterious effect on spore delivery. This belief was based on the fact That Other vascular collapsing agents, Such As dimethylxanthenone-5.6-4-acetic acid and combretastatin A-4, s been Shown to Exert Their effects in combination with radioactively labeled antibodies Only When Administered after, and not before, the anti-bodies (34, 35). Several obstacles in pubs before COBALT realistically Can Be Considered for clinical trials. For example, the range of tumors COBALT Which Might Be Addressed successful HAS not yet leg. Although the first two tumor models we Investigated (xenografts of HCT116 colorectal cancer cells and syngeneic B16 melanoma cells) effectively Were Treated, preliminary experiments show Equally Thats not all tumors are susceptible to COBALT. Greater understanding of the basis for C. Novyi-NT tumor cell killing Should Provide Insights into this issue. Additionally, differential tumor types May differential requirement chemo therapeutic agents to Achieve maximum effect in combination with C. Novy-NT. Another issue concerns the size of the tumors to be Treated. Theymust be large enough to have outgrown Their blood supply containerization and necrotic regions. We do not believe this factor limiting for many Will Be human tumor situation, as the great Majority of Clinically apparent human tumors containerization large necrotic regions (Fig. 1). However, micrometastatic disease static Might not be suit-able for COBALT. At the Other end of the spectrum, COBALT-mediated lysis of large tumors was associated with significant toxicity. Although the basis for this toxicity is not yet Known, it Could Have Been due to efflux of toxic bacterial products from the tumors or due to "tumor lysis syndrome." It HAS Previously noted thats the rapid bone lysis or very large tumor burdens is associated with systemic toxicity in humans Treated with chemotherapy, Perhaps due to the sudden efflux of tumor cell metabolites, Such As calcium, phosphate, and Uric acid, enter the circulation (36). Although tumor lysis syndrome can-be controlled in humans, it is Difficult to control in mice. Any therapy Which May Be Dramatically shrinks tumors subject to this side effect. Encouraging One of the observations made in our study is That a subset or Completely regressed tumors after a single dose of COBALT here. Additionally, the C. Novyi-NT spores Themselves Were Completely nontoxic to normal mice. The concept COBALT Could Easily be extended and improved, with differential chemo therapeutic agents or combinations to suit Particular tumor types. Additionally, it May Be Possible to genetically Manipulating the C. Novyi-NT to Enhance Their potency or selectivity. Further studies along theses lines Seem warranted.

Jain and Forber criticize the COBALT study and seems sensible to read.

Can engineered bacteria help control cancer?
Rakesh K. Jain * and Neil S. Forbes

Edwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114

* To Whom reprint requests Should Be Addressed. E-mail: jain@steele.mgh.harvard.edu .

Hypoxia and anoxia are pathophysiologic must Characteristics of solid tumors (1, 2). For Nearly 150 years, nonpathogenic, anaerobic bacteria That preferentially localize and proliferate in the hypoxic regions of tumors have leg Investigated as treatments for experimental and human tumors with mixed success (Table 1). In recent years, there Has Been A renewed interest in using bacteria thesis as innovative delivery vehicles for gene therapy (Table 1). Now, as described in this issue of PNAS, Vogelstein and coworkers (11) have created a new strain of anaerobic bacteria, devoid toxic genes or ITS, That leads to dramatic and prolonged regression of tumors Subcutaneous systematically When Administered with Conventional drugs. This strategy, Referred to as combination bacteriolytic therapy (COBALT), adds a new weapon in the war against cancer. However, there are still obstacles That Need to be over come before it Can Be Used Safely in the clinic. Ironically, a tumor's metabolically compromised micro-environment Provides a port for a number of anaerobic bacteria. In tumors, blood vessels are structurally and functionally abnormal, tion results in tempo rally and spatially heterogeneous blood flow (19, 20). This heterogeneity of nuisances the delivery of blood-borne therapeutics to all cancer cells and leads to acutely and / or chronically hypoxic and acidic regions in tumors (Fig. 1). These conditions reducing the effectiveness of radiation and some chemotherapy agents and therapeutic selective for cancer cells That are more aggressive, meta-static, and resistant to therapies Various Artists (2, 21). Ironically, a tumor's metabolically compromised micro-environment Provides a port for a number of anaerobic bacteria. And indeed, over the Past 50 Years, Several strains of facultative and obligate anaerobic bacteria have leg Shown to localize and cause lysis in transplanted tumors in animals (Table 1). These initial animal studies were so Encouraging Clinical Trials Using Clostridium That Began in the 1960s (8). Unfortunately, the results Were not as impressive as anticipated and the trials Were discontinued. So why is there a Resurgence of interest in using bacteria to treat solid tumors? To answer this question we need to Examine the criteria for an ideal anti-cancer bacterium. They Should Be: (i) nontoxic to the host, (ii) Able to replicate only within-the tumor, (iii) motile and bootable to disperse evenly throughout a tumor (include hypoxic and necrotic regions), (iv) and slowly eliminated Completely from the host (v) nonimmunogenic, and (vi) Able to cause lysis of tumor cells by direct competition for nutrients, localized production of cytotoxins, or production or therapeutic amplifiers. In the last decade, significant progress Has Been Made onEach thesis or fronts. Multiple approaches have leg Used to remove the toxin genes of bacteria (16, 17). For instance, Dang et al (1911) used heat shock to Eliminate the lethal toxin genes from Clostridium Novyi, located within-a phage episome. Modern molecular approaches Might Be Used once or genomic sequences Various strains of bacteria become available (22, 23). Of course, the use NaturallySpeaking or nonpathogenic bacteria (eg, Clostridium oncolyticum) Might Avoid the toxicity problem altogether. Additionally, Techniques Developed to transfer genetic material Other Than Writing bacteria Escherichia coli, for example the anaerobic bacteria Clostridium acetobutylicum (24) and Bifidobacterium longum (25), have the potential to modulate the toxicity, Motility, and protein expression of therapeutic bacteria. Currently there are no rapid, reliable, and inexpensive methods to screen for an ideal bacterium. Dang et al (11) screened 26 strains of bacteria for Their ability to spread evenly throughout poorly vascularized regions of tumors. The selected bacteria Were seen growing throughout the enlarged necrotic regions of tumors after systemic injection of spores. Apparently, the bacteria Were Destroy the viable cells at the interface of the necrotic region, and Using the degradation products as nutrients. However, this treatment did not Completely Eradicate the tumor, leaving a ring of viable tumor cells at the periphery. To kill viable cells in the ring, Dang et al chose to combine the bacteriolytic therapy with low molecular weight Conventional chemotherapy (mitomycin C and cytoxan). Their rationale was That Would the bacteria lyse the tumors from the inside out, and low molecular weight chemotherapeutic agents, therapeutic Would attack cancer cells in the well-perfused, non-necrotic region, a concept Used since 1964 (7) (Table 1). To Enhance the effects of chemotherapeutics (mitomycin C and cytoxan) and bacteria, Dang et al Used dolastatin (D-10), an anti-vascular agent. To our knowledge, this is the first time anti-vascular therapy with Has Been combined bacteriolytic therapy. The addition under this benefit or to COBALT, as described by Dang et al, Is That vascular stasis increases the Extent of hypoxia thereby Increasing the size of the region, affected by C. Novyi. It Appears That this combination is the predominant reason for the effectiveness of COBALT. A problem with the low molecular weight chemotherapeutics Is That They Are Rapidly cleared from perfused regions (ie, the viable ring) (26). Including the Additional benefit of anti-vascular agents lead to vascular shutdown That Is That They CAN kick extravasated molecules in tumors (27), thereby enhancing exposure to therapeutic agents in combination therapy. Indeed, COBALT therapy did produce impressive results. Dang et al Treated Two Different tumor lines grown subcutaneously in mice and observed regression in tumors and must complete a task in recitals or printable proportioning That mice survived. Whethere similar cure rates Can Be Achieved with Cobalt in orthotopic and spontaneous tumors needs to be Examined. COBALT Besides, there are Several Other Strategies That Amplify bacteriolytic therapy. One or thesis is to engineer bacteria to produce inflammatory cytokines (eg, tumor necrosis factor α) That Increase the sensitivity of tumors to radiation therapy and / or Evoke a host immune response (28). Another approach is bacteria-directed enzyme prodrug therapy (BDEPT), a variation or antibody-directed enzyme prodrug therapy (ADEPT). In this approach, targeted bacteria are engineered to produce enzymes activate prodrugs That can-within-the tumor (9, 29). Another Possibility is to place the genes or prodrug-activating enzymes under the control of radiation-inducible promoters to providence spatial and temporal control, Galanthus enabling selective killing of tumor cells while sparing normal cells (28, 30). So what are the potential problems with bacteriolytic therapy? First, there is the immediate problem Encountered by Dang et al: toxicity. Just after Removing the toxin genes, COBALT therapy led to ~ 15-45% mortality in mice. Whethere this is caused by the so-called tumor lysis syndrome (31) or the efflux of toxic bacterial products is Not known. Identification of the toxins released by lysing Rapidly tumors or by large colonies of Clostridium contained within-tumors is essential for alleviating the toxicity. Overexpressed Toxins May Be Identified by the bacteria after complete sequencing of the respective genomes. Well-known strategies then Can Be Used To tackle specific toxins. On the otherhand, alleviating the toxicity from low molecular weight byproduct of dying cells-meaning requirement Careful control of the rate of tumor lysis. Once the issues of systemic toxicity and incomplete tumor lysis are Addressed, there are potential pitfalls That Other May impedance the success of COBALT therapy in the clinic. The must thesis is significant or acquired drug resistance, Which Lowersthe effectiveness of the standard chemotherapeutics Used in COBALT after repeated treatment. Just Such As new drug Gleevec are facing this age-old problem (32). However, antiangiogenic and anti-vascular agents May Be less susceptible to this type of resistance (21, 33). Combined bacteriolytic anti-angiogenesis therapy (COMBAT) May, Galanthus, or come to Circumvent the problem of drug resistance. A third and more Difficult Problem Is That or treating small non-necrotic metastases or large primary tumors. The current strategy is to treat metastases as early as Possible with Conventional chemotherapeutics before the onset of physiological and / or multidrug resistance. COBALT Would Require one to Wait Until the developement metastases hypoxic / necrotic regions. Because metastasis is the major cause of mortality from cancer (34), we wonder whethere Would it be Possible to engineer bacteria That can-localize in small orthotopic tumors and spontaneous metastases That Their containerization large hypoxic regions do not? Such bacteria Would Facilitate not only treatment of metastases but Also Their early detection by Using molecular imaging techniques. References and adresses for more information at this page inPubMed