Quantified color Doppler sonography of tumor vascularity in an animal model.

This study was designed to evaluate the accuracy of a system to quantitate tumor vascularity with amplitude (power) color Doppler sonography two- and three-dimensionally. The vascularity of 20 transplanted murine tumors was determined with quantitated amplitude color Doppler sonography both two- and three-dimensionally and compared to tumor vascularity estimated by histologic examination. Serial examinations were performed 15, 30, 45, and 60 min after the injection of the exotoxin CM-101 and saline solution to assess changes in tumor vascularity. Three-dimensional amplitude color Doppler sonography best depicted the overall vascularity of tumor when compared to histologic estimation of vessel density. However, neither two- nor three-dimensional amplitude color power angiography correlated well to the microvessel count, probably a reflection of the difference in the method for vessel quantification using sonographic versus histologic techniques. Three-dimensional amplitude Doppler sonography correlated better with counts of large vessels (> 100 microm) as opposed to small vessels (> 15 microm). Time-activity curves showed no difference in tumor flow at the times measured in the experimental group injected with CM-101 or when compared to saline solutions in either the peripheral or central portions of the tumor. This three-dimensional amplitude color Doppler sonographic system affords global quantification of tumor vascularity and flow that may, in turn, be useful in determining the probability of malignancy (by determination of branching patterns and vessel regularity) or tumor response or both to treatment.

Does age of the host affect growth rates of skeletal malignancies?

Statistical analysis of bone tumor growth rates as a function of age at initiation of radiation-induced skeletal malignancies in our animal colony indicated that the p value for an association between these parameters was <0.05, suggesting a correlation in beagle dogs. The youngest animals appeared to exhibit the most slowly growing tumors, and the trend was toward more rapidly growing tumors with increasing age. Less effective immune systems in older animals were invoked as a possible explanation of this relationship.

Functional studies on the anti-pathoangiogenic properties of CM101.

Group B streptococcus (GBS) isolated from human neonates diagnosed with sepsis and respiratory distress produces a polysaccharide exotoxin (CM101) which has been previously described as GBS toxin. CM101 infused i.v. into tumor-bearing mice causes rapid tumor neovascularitis, infiltration of inflammatory cells, inhibition of tumor growth and tumor apoptosis. CM101 has successfully completed phase I studies in refractory cancer patients with very encouraging results. We have now demonstrated a mechanism of action for CM101. Using a normal mouse tumor model, we have examined tumor and normal tissues which were harvested at 0, 5, 15, 30 and 60min post-infusion of either CM101 or dextran. We present evidence that CM101 is rapidly (within the first 5min) bound to the tumor neovasculature. Complement is activated by the alternative pathway (C3) and leukocytes start to infiltrate the tumor within the first 5min. Through RT-PCR and immunohistochemical techniques, we demonstrate that proinflammatory cytokines, interleukin-6 and tumor necrosis factor (TNF)-alpha, are up-regulated in infiltrating leukocytes and TNF receptor 2 is up- regulated in the targeted tumor neovasculature. Combined, these events constitute possible explanations for the observed pathophysiology of tumor ablation.

Phase I study of the antineovascularization drug CM101.

CM101 is a bacterial polysaccharide that induces neovascular inflammation in malignant tumors. Fifteen patients with refractory malignancies received CM101 i.v. by a 15-min infusion every other day, three times in 1 week, at doses ranging from 1 unit (7.5 microgram)/kg to 5 units/kg. Serum was analyzed for anti-CM101 IgG and IgM weekly. Plasma levels of inflammatory cytokines, including tumor necrosis factor alpha, interleukin 8, interleukin 10, MIP-1alpha, and soluble E-selectin, were analyzed from -15 min to 12 h during each treatment. Dose-limiting toxicities, including grade IV dyspnea and arrhythmia, were encountered at the 5-unit/kg level. Toxicities occurred primarily within the first 12 h after therapy and included mild-to-moderate fever and chills, nausea, cough, headache, facial flushing, dyspnea, myalgias, and acute tumor-related pain. No patient developed detectable antibodies to CM101. All patients experienced marked time- and dose-dependent elevations in all cytokines studied. Three patients experienced tumor shrinkage. The results show that CM101 can be safely administered at doses that produce evidence for severe, and possibly tumor-specific, inflammation. Further study is necessary to better characterize the mechanism of action and determine the optimal dose and schedule of this new agent.

Soluble E-selectin in cancer patients as a marker of the therapeutic efficacy of CM101, a tumor-inhibiting anti-neovascularization agent, evaluated in phase I clinical trail.

A polysaccharide toxin, GBS toxin, is produced by group B Streptococcus (GBS) isolates from neonates who died of "early-onset disease". GBS toxin, named CM101 in the clinic, was hypothesized, on the basis of our previous in vivo studies, to induce inflammation in pulmonary neovasculature in neonates by cross-linking of embryonic receptors still expressed after birth and in tumor neovasculature in adults. Immunohisto chemical in vitro analysis of human biopsies showed that tumor neovasculature is indeed a binding site for CM101. In vivo studies in mice have demonstrated that CM101 induced inflammatory responses in neoplastic tumor neovasculature causing inhibition of tumor growth and tumor cell necrosis. These experimental observations warranted a phase I clinical trial for CM101 as an anti-neovascularization agent in human cancer therapy. Cancer patients received one cycle of therapy consisting of three treatments during 1 week. CM101 was administered over 15 min by i.v. infusion. Dosages of 7.5 micrograms/kg (1 U/kg), n = 3; 15 micrograms/kg (2 U/kg), n = 6; 24.75 micrograms/kg (3.3 U/kg), n = 3; and 37.5 micrograms/kg (5 U/kg), n = 3 were used. Enzyme-linked immunosorbent sandwich assays (ELISA) of the patients sera showed a marked elevation of soluble E-selectin with a peak concentration observed at 8-12 h after each CM101 infusion. The average baseline value for soluble E-selectin prior to the first treatment was 97.3 +/- 23.4 ng/ml (mean +/- SEM, n = 15) and the average peak level at 8 h was 441.6 +/- 62.4 (mean +/- SEM, n = 15; P < 0.001). Subsequent treatments gave average maximum soluble E-selectin levels again at 8 h of 466.9 +/- 87.6 and 412.0 +/- 67.8 ng/ml, for treatments 2 and 3 respectively. Baseline values for treatments 2 and 3 were 192.3 +/- 26.4 and 226.4 +/- 26.1 ng/ml respectively (p < 0.01 versus treatment 1). Out of 15 patients, 5 showed tumor reduction or stabilization and were given additional cycles of therapy. CM101 induced an increase in soluble E-selectin levels, which remained elevated over baseline at the start of the following treatment cycles. The baseline remained elevated for several weeks after the final treatment, i.e., P < 0.01 for levels before treatment 1 compared to those at week 4 after treatment. Elevated soluble E-selectin is considered proof of endothelial engagement in an inflammatory process. Our data support the contention that the inflammatory response observed in these cancer patients is targeting the tumor neovasculature and that measurement of soluble E-selectin levels in patients treated with CM101 can provide important information on the magnitude of CM101-mediated neovascular endothelial activation and tumor cell damage in cancer of endothelial origin, or cancer with a major neo-angiogenic component.

Quantification of tumor vascularity and flow with amplitude color Doppler sonography in an experimental model: preliminary results.

This study was designed to evaluate a system to quantitate vascularity and tumor blood flow with amplitude (power) color Doppler sonography. The vascularity of nine transplanted murine tumors was determined with quantitated amplitude color Doppler sonography and compared to tumor vascularity estimated by histologic examination. The system used seemed to provide an accurate depiction of the vascularity of tumor vis-àa-vis histologic estimation of vessel density (r = 0.80). Time-activity curves showed greater flow in the experimental group injected with an exotoxin than in the group injected with saline solution. Vascular density quantification with amplitude color Doppler sonography also was more accurate when an intravascular agent (such as an exotoxin) was used than when saline infusions were given. This quantification scheme may allow the development of a system to assess the probability of malignancy and to monitor tumor response to treatment on the basis of the vascularity of the mass.

Acute inflammatory changes in subcutaneous microtumors in the ears of mice induced by intravenous CM101 (GBS toxin).

CM101, a bacterial polysaccharide derived from group B streptococcus, induces pronounced inflammatory changes in and around tumor blood vessels 60 min after i.v. injection. A technique has been developed for implanting small numbers of tumor cells in the ear skin of mice. This allows macroscopic examination of the tumor and its supporting blood vessels as it reaches the 10000 cell size and greater. Treatments can be monitored in this model for effects on small "metastatic-like" tumor nodules by direct observation and by histological examination. Inflammatory changes were indicated by increased numbers of polymorphonuclear leukocytes (PMN) adjacent to and marginating within thin-walled blood vessels and within the tumor tissue. PMN were seen in the process of migrating through venules and enlarged capillaries, each with prominent endothelial cells. Tumor morphology was variable with evidence of occasional single necrotic cells. This contrasted with tumors in ears of dextran-treated or untreated mice, which had uniform tumor morphology, and acute inflammatory cells were rarely present.

CM101, a polysaccharide antitumor agent, does not inhibit wound healing in murine models.

CM101 (previously called GBS toxin), a new anticancer polysaccharide that induces inflammatory reactions in neovasculature of tumors, does not cause similar reactions in neovasculature of healing wounds. It appears that treatment with CM101 will not interfere with normal wound healing in cancer patients.

Occurrence of metastases in beagles with skeletal malignancies induced by internal irradiation.

Metastases from malignant bone tumors often are responsible for the fatal effects of these cancers. Characteristics of primary skeletal malignancies in beagles injected with bone-seeking radionuclides were studied by Thurman (1971) and summarized by Thurman et al. (1971). There were 212 tumors in 186 of these dogs for which we subsequently received information on bone tumor metastases. Evaluation of bone and soft tissue slides from these animals allowed us to compare parameters reported previously with the occurrence of grossly apparent bone tumor metastases. Data included growth-rate of the primary tumor, volume of the primary tumor at death, sex of the animal, growth period of the primary tumor, degree of calcification of the primary tumor, skeletal location of the primary tumor, cumulative radiation dose to the skeleton, dose equivalent to the skeleton, and year of death. For most of the comparisons, no significant differences could be established between dogs with and without metastases. However, tumor volume at death appeared to be correlated with probability of metastasis (p < 0.05), with the larger tumors being associated with higher rates of metastasis. Comparisons of dogs with and without metastases as a function of tumor growth-rate did not, for the most part, yield significantly different results between groups. Exceptions were when only one tumor per dog was considered for animals with multiple primary tumors and when only the tumor with the longest doubling time was included for all dogs with multiple primary tumors (p < 0.02). This effect was a result of only two tumors with doubling times > 45 d. Both had been characterized by Thurman (1971) as among the tumors with the least uncertainty in calculated doubling times. Rates of metastasis in dogs with primary tumors in paired bones were significantly higher than corresponding values of dogs with primary tumors in unpaired bones. Metastases in dogs with primary tumors in the ribs appeared to be more pronounced and those in the thoracic vertebrae appeared to be less pronounced than for animals with primary tumors in other bones as compared with the average for the whole skeleton.

Effects of group B Streptococcus toxin on long-term survival of mice bearing transplanted Madison lung tumors.

GBS toxin is a polysaccharide exotoxin produced by group B Streptococcus. This organism causes sepsis and respiratory distress in human neonates (so-called early onset disease). This disease is marked by a strong inflammatory response only in the lung, with pulmonary sequestration of granulocytes and extensive capillary endothelial damage, and occurs only during the first few days after birth. We have found that a similar inflammatory response can be induced by i.v. infusion of picomole quantities of GBS toxin in the developing vasculature of transplanted tumors in mice and can significantly retard the tumor growth. When optimum treatment with GBS toxin was started shortly after tumor implantation, a majority of tumors in the mice regressed and the mice remained tumor-free for over 5 months. Some tumors regressed in mice receiving short-term treatment with GBS toxin, but recurred after the treatment was stopped. Median survival times were extended by all regimens and all doses of GBS toxin tested. No evidence of toxicity to the vasculature of other tissues was observed. GBS toxin is being tested for cancer therapy in humans.

Antitumor effects of GBS toxin: a polysaccharide exotoxin from group B beta-hemolytic streptococcus.

A group B streptococcus (GBS) isolated from human neonates diagnosed with sepsis and respiratory distress ("early-onset disease") produces a polysaccharide exotoxin (GBS toxin) that, when infused in sheep, causes lung pathophysiology similar to that seen in humans. Histological studies have demonstrated that GBS toxin induces a strong inflammatory response in the lung, with pulmonary sequestration of granulocytes and extensive capillary endothelial damage. The susceptibility of humans to GBS toxin is age-dependent and limited to about 4 days after birth. It is rarely evident thereafter. This suggests that the binding of GBS toxin to the target endothelium occurs via specific components in the developing lung endothelial cells of the newborn that are later lost. We report here that GBS toxin can also bind to developing endothelium associated with neoplasia and induce an inflammatory response. GBS toxin was shown by immunohistochemistry to bind to capillary endothelium of human large-cell carcinomas. In nude mice bearing human tumor xenografts, intravenously administered GBS toxin caused tumor necrosis and hemorrhagic lesions, and substantially inhibited the rate of growth of the tumors. In BALB/c mice bearing Madison lung tumors, GBS toxin induced an inflammatory response resulting in marked changes in tumor morphology, including vasodilation, endothelial and tumor cell necrosis, invasion of lymphocytes and macrophages, and capillary thrombosis. In these tumor models, no evidence of toxicity to the vasculature of other tissues was observed. The reported pathophysiology of GBS in human neonates, the lack of disease in non-neonates colonized with GBS, and these results suggest that GBS toxin may have potential as a well tolerated agent in cancer therapy of some human tumors.

Cultured cell lines from human breast cancer biopsies and xenografts.

Eighty-five breast cancer specimens were processed as part of a program in tumor acquisition, propagation, and preservation for biotherapy. Nine long-term culture cell lines were developed. Four cell lines were from solid tumor metastases, two lines were from pleural fluid specimens, and three were from xenograft tumors grown in nude mice. Two of the xenograft-derived cell lines were from biopsies which produced tumor cell lines as well. Success in establishing cultures did not correlate with the viability of the biopsy received. Poor tumor cell attachment to culture plastic was the most common problem. For certain specimens, attachment and growth were enhanced on collagen and extracellular matrix substrates. Collagen was beneficial in the development of one cell line. The cell lines were characterized and each of the lines contained more nuclear DNA than found in normal cells. Four of five lines tested were tumorigenic in nude mice. Five of nine were clonogenic in soft agar. Each of the cell lines tested reacted with at least two anti-tumor monoclonal antibodies. Xenograft and biopsy-derived cell lines from the same tumor were similar in their characteristics. While breast cancers are indeed difficult to establish and propagate in culture, the use of xenografts and special substrates appears to be beneficial in the development of cell lines from some tumors.

Cultured breast cystosarcoma phylloides cells and applications to patient therapy.

Malignant cystosarcoma phylloides (CP) is a relatively rare cancer of the breast. A CP tumor was processed as part of a tumor acquisition, propagation, and preservation program in patient biotherapy. Two tissue culture cell lines were developed from this tumor, one directly from the biopsy, another from a xenograft tumor grown in athymic mice. The two cell lines were similar in character. There was strong immunochemical reactivity with antibodies to vimentin, type I collagen, and type III collagen. There was no reactivity with antibodies to cytokeratin and epithelial membrane antigen. Both cell lines were aneuploid, clonogenic in soft agar, and tumorigenic in nude mice. 5 alpha-dihydrotestosterone and thyroxine added to the culture medium stimulated growth, while testosterone, 17 beta-estradiol, and 4-hydroxytamoxifen were without effect. Dexamethasone and cortisol were inhibitory at high doses (10(-6) M). Dibutyryl cyclic AMP, theophylline, and vitamin C were all inhibitory. The biopsy contained tumor-infiltrating lymphocytes which proliferated in cultures containing interleukin 2. The expanded lymphocytes were activated T cells which had the capacity to lyse tumor cells. These results suggest possibilities in the therapy of cystosarcoma phylloides involving vitamin C, certain hormones, and tumor-infiltrating lymphocytes.

Activation and expansion of tumor-derived activated cells for therapeutic use.

We obtained tumor specimens from patients with cancer in an effort to activate and expand the tumor-infiltrating lymphocytes for therapeutic use. With the use of finely minced tumor preparations from eight different tumor types, recombinant interleukin-2, and lymphokine-activated killer cell-conditioned medium, lymphocytes were expanded in vitro. After 4 weeks, the tumor cells were virtually absent from the cultures. At this point, the lymphocytes were termed "tumor-derived activated cells" (TDACs). Over 90% of the TDACs from each of the different tumor types were T lymphocytes, and the percentage of cells expressing either CD4 or CD8 varied considerably from population to population. The lymphocytes showed specific cytolytic activity in melanoma and colon and renal cell carcinomas. Continued expansion and long-term growth of the TDACs, as well as maintenance of the cytolytic activity, were achieved by periodic stimulation of the TDACs with irradiated autologous tumor cells. In a clinical study of 28 patients with cancer, we generated a mean number of 1.2 X 10(11) TDACs in an average time in culture of 69 days. These TDACs were subsequently infused into the patients with cancer. TDACs appear to represent an important resource for biotherapy of patients with cancer.

Tumor acquisition, propagation, and preservation. The culture of human colorectal cancer.

Fourteen new colorectal cancer cell lines were developed as part of a tumor acquisition, propagation, and preservation program for biotherapy. Fifty-six specimens were received. Nine cell lines were generated from biopsies; seven of these cell lines were from metastatic lesions. Five additional cell lines were developed from xenografts grown in nude mice. Biopsies that produced three of these xenografts gave rise to parallel culture cell lines. Biopsy-derived and xenograft-derived cell lines from the same tumor behaved similarly in culture and exhibited similar markers when assessed immunohistochemically. Collagen substrate was beneficial in the primary culture of 50% of the specimens tested. Collagen was required for the successful propagation of two cell lines.

Immunoconjugates of doxorubicin and murine antihuman breast carcinoma monoclonal antibodies prepared via an N-hydroxysuccinimide active ester intermediate of cis-aconityl-doxorubicin: preparation and in vitro cytotoxicity.

Doxorubicin (DXR) conjugated to murine monoclonal antibodies (MoAb) raised against human breast tumor cells demonstrated a MoAb-specific, molar ratio-dependent in vitro cytotoxicity. These conjugates were prepared on a scale sufficient to allow for subsequent clinical trials (1 to 3 g of MoAb per conjugation reaction). The conjugation reaction proceeded via an N-hydroxysuccinimide (NHS) active ester intermediate of cis-aconityl-DXR (CA-DXR), resulting in a cis-aconitate acid-sensitive linker between the DXR and MoAb. Molar ratios of DXR to MoAb ranged from 40 to 45. The immunoreactivity of conjugated MoAb was only slightly decreased from naked MoAb. When immunoconjugates were incubated with MoAb-reactive tumor cells for 3 hours, specific cell-killing was observed. If the exposure time was lengthened to 18 hours, however, nonspecific killing resulted. Incubation of the immunoconjugate with the nonspecific adsorbant Amberlite XAD-2 caused an average 30% decrease in the DXR-to-MoAb molar ratio, suggesting a population of drug that is tightly but noncovalently associated with MoAb.

Chemosensitivity testing in a tumor acquisition, propagation, and preservation program.

Chemosensitivity assays were carried out as part of a tumor acquisition, propagation, and preservation program for cancer biotherapy. In addition to biopsy specimens, tumor cells propagated in culture or tumor xenografts grown in nude mice were submitted for chemosensitivity assay when sufficient biopsy material was unavailable. Chemosensitivity was tested utilizing the adhesive tumor cell culture system. A total of 154 specimens was submitted for testing; 96 specimens were assayed. Success rates were 55% for primary cancer biopsies, 67% for metastases, 69% for xenografts, and 70% for cell lines. There were no significant differences evident when the sensitivity to drugs of tumor cells originating from biopsies, xenografts, or tissue culture were compared. Sufficient data were available for 18 patients to compare clinical results of drug treatment with predictive results from the chemo-sensitivity assay. Assay results indicating insensitivity appeared to predict resistance; however, assays indicating sensitivity were not predictive. These results suggest that propagated tumor material, such as xenografts and cultured cell lines, may be useful when biopsy tissue is not available.

Induction of surface marker changes and monoclonal idiotypic immunoglobulin secretion in lymphoma/leukemia cells: comparative study with interleukin-1, interleukin-2, interleukin-4, 8-bromo-guanosine, pokeweed mitogen, and tetradecanoyl phorbol-13-acetate.

Induction of differentiation in B lymphoma/leukemia cells with interleukins was compared with differentiation induced by phorbol ester (TPA) and pokeweed mitogen (PWM) or by 8-bromo-guanosine. Both cell surface changes and monoclonal immunoglobulin (Ig) secretion were followed as markers of differentiation. The results indicate great similarity in the differentiation patterns induced by interleukin-1 (IL-1), interleukin-2 (IL-2), and interleukin-4 (IL-4), with regard to Ig secretion and changes in surface markers. Induction of Ig secretion and surface marker changes by 8-bromo-guanosine was similar to that induced by TPA and PWM; however, for some markers, cell surface changes induced by TPA and PWM or by 8-bromo-guanosine were quite different from those induced by the three interleukins tested. Whereas all three interleukins stimulated the expression of CD5, PWM and TPA and 8-bromo-guanosine substantially decreased CD5 expression on B lymphoma cells. Differences were also observed in the effect on the expression of surface Ig and on the expression of CD19 and CD20. Interestingly, the three interleukins tested and 8-bromo-guanosine induced differentiation and Ig secretion within 24 to 48 hours with no prior activation by B-cell activators, such as anti-surface Ig antibody. These results suggest that leukemic B cells are arrested at a point distal to activation and first cell division. Moreover, the similarity in Ig secretion and surface changes induced by TPA and PWM or 8-bromo-guanosine suggest a similar pathway; however, this pathway is different from the differentiation signal induced by the three interleukins.

Enhancement of human lymphokine-activated killer cell cytolysis and a method for increasing lymphokine-activated killer cell yields to cancer patients.

In a continued effort to make interleukin-2/lymphocyte-activated killer (LAK) cell therapy safer and more efficacious for cancer patients, we examined methods of increasing the yields of cells obtained as a final product for reinfusion. Previously, the major cell loss occurred in the Ficoll-Hypaque gradient separation procedure used before cell culture. Therefore, we investigated the necessity of this step. Leukapheresis procedures (n = 105) from 40 different cancer patients showed that the resultant cell product is predominantly mononuclear (lymphocytes and monocytes; greater than 97%) before the gradient purification step. The only cells observed to decrease in percentage as a result of the step were red blood cells (RBC: WBC ratio of 17:1 before purification to 1:3 after purification). Our study showed that the cytolytic potential of unpurified leukapheresis products against the LAK-sensitive line Daudi and the natural killer cell-sensitive line K562 was greater and that the patients received significantly more cells at times of reinfusion if the gradient separation step was eliminated. By additional experiments, we determined that autologous red blood cells enhance the generation of cytolytic LAK cells. This enhancement was greater if the red blood cells were in contact with the mononuclear cells during the time of cell culture. The elimination of the Ficoll-Hypaque purification step not only reduces the time and cost of the cell collection procedures, it also allows us to return to the patients greater numbers of cytolytic LAK cells following the activation period.

Characterization of a method using viable human target cells as the solid phase in a cell concentration fluorescence immunoassay (CCFIA) for screening of monoclonal antibodies and hybridoma supernatants.

A new method has been characterized for the use of viable target cells as the solid phase for screening of hybridoma supernatants in a cell concentration fluorescence immunoassay (CCFIA). Briefly, the specific target antigen on the cells is bound by the monoclonal antibodies and revealed by use of a fluoresceinated second antibody. Separation of free from bound antibody is accomplished by filtration in the 0.2 micron filter-bottom wells of specialized assay plates. Processing is automated in a Pandex screen machine, resulting in numerical fluorescence values for each well. This method is rapid (under 1 h per 96-well plate), highly sensitive (down to 0.2 ng/ml) and sparing of target cells (0.3-2.5 X 10(4) cells per assay well). It has been applied to 37 different varieties of human solid tumor cells, as well as to human peripheral blood mononuclear cells. The cells used as targets for the characterization of this method were still capable of attachment and growth when recovered post-assay. This method was compared with a viable cell enzyme-linked immunosorbent assay (ELISA) method, showing similar sensitivity and greatly shortened assay time. Comparison of the results from this method with those obtained from flow cytometric analysis performed on viable cells showed close correlation, whereas a lower correlation was seen with immunohistochemical methods using acetone-fixed cells. Development of this method made it possible to rapidly screen many thousands of hybridoma supernatants and successfully select those which were specific for surface antigens on viable cells.