Barbara D. Wamil

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 stimulates cutaneous wound healing through an anti-angiogenic mechanism

CM101, an anti-pathoangiogenic polysaccharide derived from group B streptococcus, has been shown to inhibit inflammatory angiogenesis and accelerate wound healing in a mouse model and minimize scarring/gliosis following spinal cord injury. To evaluate the in vivo effects of CM101 on cutaneous wound healing in the pig, intravenously delivered CM101 or placebo vehicle was given 1 h after cutaneous wounding and again at 72 h after injury. Tissues from partial-thickness and full-thickness excisions were collected at days 4 and 7 after wounding and evaluated for a variety of standard healing parameters. Both types of CM101-treated wounds showed significantly less evidence of inflammatory angiogenesis when assessed by macroscopic photography of the wound surface, qualitative histological observations, laser doppler perfusion imaging, and quantitative morphometric analysis of microvessel area from endothelium selectively immunostained for factor VIII. Resurfacing was accelerated in partial-thickness and full-thickness excisions that received two doses of CM101 as compared to the placebo-treated excisional wounds. Neodermal thickness was increased in CM101-treated wounds at day 4 and was slightly reduced in comparison with placebo by day 7. New collagen accumulation appeared to be unaffected by the CM101 treatment. Immunohistochemical staining using a polyclonal antisera directed against the anti-pathoangiogenic CM101 target protein HP59 on day 7 indicated a strong immunoreactivity on the microvessels present in the control wounds but not in wounds of the CM101-treated animals. In summary, the immunolocalization HP59 in the microvessels of the cutaneous wound bed in control but not in CM101 treated wounds suggests that CM101 inhibits the pathologic inflammatory angiogenesis accompanying the normal granulation processes. The net biological effect of inhibited inflammatory pathoangiogenesis is a diminished, suggested and purely physiologic, microvascular bed which translates into an enhanced rate of epithelial resurfacing and therefore an overall accelerated rate of wound repair.

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 60 min post-infusion of either CM101 or dextran. We present evidence that CM101 is rapidly (within the first 5 min) bound to the tumor neovasculature. Complement is activated by the alternative pathway (C3) and leukocytes start to infiltrate the tumor within the first 5 min. Through RT-PCR and immunohistochemical techniques, we demonstrate that proinflammatory cytokines, interleukin-6 and tumor necrosis factor (TNF)-α, 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.