Kimberly Credit

Intraperitoneal immunotherapy for metastatic ovarian carcinoma : Resistance of intratumoral collagen to antibody penetration

Purpose: Convective transport of macromolecules from the peritoneal cavity into tumor is determined by its hydraulic permeability and the pressure gradient. Previous studies showed that establishing a pressure gradient into the tumor failed to result in significant penetration. This study addresses the hypothesis that the extracellular matrix is the major resistance to the penetration of an i.p. injected antibody. Experimental Design: Human ovarian tumors (SKOV-3 and OVCAR-3) were established in the abdominal wall of athymic rats. After anesthesia, the tumor serosal surface was treated for 2 hours with Krebs solution (control), collagenase (37.5 unit/mL), or hyaluronidase (10 unit/mL) followed by 3 hours of convective delivery of radiolabeled IgG. Transport of antibody into the tumor was measured with quantitative autoradiography along with the tumor interstitial pressure, concentration of collagen and hyaluronic acid, and IgG volume of distribution. Results: Antibody was excluded from 42% to 53% of tumor extracellular volume. Exposure of tumors to hyaluronidase did not enhance IgG transport despite removal of 90% of the hyaluronan from the exposed tumor. In contrast, collagenase reduced collagen content, lowered tumor interstitial pressure, and markedly enhanced antibody penetration. Conclusions: Reduction of collagen, but not hyaluronan, in the matrix of ovarian xenografts enhanced the transport of i.p. injected antibody. Although high interstitial pressure is a deterrent to convective transport of macromolecules into the tumor parenchyma, the structure of the interstitial matrix provides an inherent resistance, which must be overcome before effective delivery of an antibody.

Resistance of Tumor Interstitial Pressure to the Penetration of Intraperitoneally Delivered Antibodies into Metastatic Ovarian Tumors

Purpose: Despite evidence that regional chemotherapy improves the treatment of metastatic peritoneal ovarian carcinoma, monoclonal antibodies have not shown significant success in i.p. delivery. The present study was designed to address the hypothesis that convective penetration of macromolecular antineoplastic agents depends on a positive pressure difference between the i.p. therapeutic solution and the tumor. Experimental Design: Nude rats with human ovarian xenografts implanted in the abdominal wall were used in experiments to facilitate in vivo measurement of tumor pressure and the treatment of the tumor with i.p. solutions at high pressures. Penetration of 125I-labeled trastuzumab was measured with quantitative autoradiography. Results: Tumor pressure profiles showed peak pressures of 32 mm Hg with mean pressures (± SD, mm Hg) in 12 SKOV3 tumors of 9.7 ± 8.3 and in 15 OVCAR3 tumors of 12.5 ± 7.0. I.p. therapeutic dwells at 6 to 8 mm Hg (maximum feasible pressure) showed significantly less penetration of trastuzumab than in adjacent normal muscle. To establish a driving force for convection into the tumor, various maneuvers were attempted to reduce tumor pressure, including treatment with taxanes or prostaglandin E1, elimination of tumor circulation, and removal of the tumor capsule. Tumor decapsulation decreased the pressure to zero but did not enhance the penetration of antibody. Binding to specific trastuzumab receptors on each tumor was shown to be not a significant barrier to antibody penetration. Conclusions: The results only partially support our hypothesis and imply that the microenvironment of the tumor is in itself a major barrier to delivery of charged macromolecules.

Peritoneal Changes after Exposure to Sterile Solutions by Catheter

Most current animal models that are used to study effects of long-term peritoneal exposure to dialysis solutions use an indwelling catheter for daily injections. It was hypothesized that the presence of a foreign body in the peritoneal cavity (PC) might alter the inflammatory response to the solutions and that the response would depend on exposure duration. For addressing these, long-term injections were carried out for 2 to 8 wk in 90 Sprague-Dawley rats: 40 via a subcutaneous port connected to a silicone catheter tunneled to the PC, 40 via direct needle injection, and 10 noninjected, age-control rats. Daily volumes were 30 to 40 ml of filter-sterilized, bicarbonate-buffered solutions that contained 4% dextrose. After 2, 4, 6, and 8 wk, anesthetized rats underwent transport experiments with a chamber affixed to the abdominal wall to determine mass transfer coefficients of mannitol (MTC(mannitol)) and albumin (MTC(BSA)), osmotic filtration flux (J(osm)), and hydrostatic pressure-driven flux. After the rats were killed, tissues were collected for measurement of peritoneal thickness, vascular density, and immunohistochemical staining. ANOVA demonstrated significant (P < 0.01) differences in thickness, vessel density, MTC(mannitol), and MTC(BSA) among the groups at the various time intervals and in overall means. Differences among the groups were less pronounced for hydrostatic pressure-driven flux and J(osm). Vessel density, MTC(mannitol), MTC(BSA), and J(osm) were dependent on injection duration (P < 0.01). There were marked differences between the needle injection and catheter injection groups at various intervals in the expression of three cytokines. It is concluded that the histologic and functional response depends on the duration of injection with animals that are exposed for as little as 2 wk demonstrating alterations. These findings confirm the hypothesis that the presence of a PC catheter increases inflammatory response to sterile solutions as evidenced by the structural and functional changes in the peritoneal barrier.

Inhibition of ovarian cancer cell metastasis by a fusion polypeptide Tat-ELP

Tumor cell metastasis is a complex, multi-step process that is a major cause of death and morbidity amongst cancer patients. Cell adhesion plays a critical role in the development of metastatic cancer, and it is mediated by interactions between receptors on the cell surface and ligands of the extracellular matrix or other surfaces. Therefore, inhibition of the cell adhesion process appears to be an effective method of preventing metastasis. This work describes a genetically engineered polypeptide with the potential to prevent cell adhesion and inhibit metastasis. We have found that the cell penetrating peptide Tat, fused with elastin-like polypeptide (ELP) inhibited adhesion, spreading, invasion and migration of SKOV-3 ovarian cancer cells in cell culture. Furthermore, we have also confirmed that Tat-ELP has anti-metastatic potential in an experimental ovarian cancer metastasis model in vivo, causing approximately 80% reduction in the tumor burden. Since cell attachment is an important step in tumor cell invasion and metastasis, these results suggest a novel role of Tat-ELP as a therapeutic intervention in cancer metastasis.

PERITONEAL INFLAMMATION AFTER TWENTY-WEEK EXPOSURE TO DIALYSIS SOLUTION: EFFECT OF SOLUTION VERSUS CATHETER–FOREIGN BODY REACTION

♦ Background: We hypothesized that both sterile solutions and foreign body reaction to the peritoneal dialysis catheter are associated with inflammatory changes in rats exposed to hypertonic solution. ♦ Methods: Four hypertonic solutions (30 – 40 mL) were injected daily via needle and syringe over 20 weeks in 4 groups of rats: 4.25% standard clinical solution (LAC), LAC plus pyridoxamine (PYR), LAC plus ethyl pyruvate (EP), and a biocompatible 4% dextrose solution (BIC). Two groups received catheters: a non-injected 4-week catheter group (C4) and a group injected for 20 weeks with the BIC solution (CI). Control animals (CON) were not injected. In the C4 group, adherent cells were separated from the catheter and examined by culture and electron microscopy to ensure that animals were bacteria free prior to exposure to solution. Animals underwent transport experiments to determine mass transfer coefficients of mannitol (MTCM) and albumin (MTCA), osmotic filtration flux (Josm), and hydrostatic pressure-driven flux (Jp). After euthanasia, tissues were examined for submesothelial thickness, vascular density, and immunohistochemistry for various cytokines. ♦ Results: The catheter cell layer was free of bacteria and consisted of macrophages, lymphocytes, mesothelial cells, and fibroblastic cells. Marked differences in angiogenesis and submesothelial thickening were noted for the catheter groups. Transport differences were mixed: MTCM was significantly less for the CI group and MTCA was variable among the groups. There were no differences among groups for Josm or Jp. Inflammatory markers in the catheter-adherent cells correlated with inflammatory changes in the tissue. These data demonstrate significant changes in submesothelial thickness, angiogenesis, transport function, and inflammatory markers between animals injected with sterile solutions over 20 weeks with and without catheters. ♦ Conclusion: An indwelling catheter amplifies peritoneal inflammation from dialysis solutions through a foreign body reaction. Our data also suggest that additives to existing solutions may have limited the effect on inflammatory response to non-biocompatible solutions.

238 ARE TRANSPERITONEAL PERMEABILITIES TO WATER AND SOLUTES EQUIVALENT FOR MICE AND RATS

Mechanistic studies of alterations in peritoneal transport are facilitated by the development of transgenic mice, but the majority of transport studies have been carried out in rats. To correlate results between species, we hypothesized that mouse transport parameters, normalized to the peritoneal contact area, would be similar to those of the rat. To address this, we affixed small ({223}10 mm dia.) plastic chambers to the serosa of the abdominal wall of anesthetized CD1 and C57bl mice. The chamber constrained transfer across the area of the chamber base and facilitated mixing, volumetric, and concentration measurements versus time for mannitol, serum albumin, and osmotic and hydrostatic pressure-driven convection. The mass transfer coefficient of mannitol (MTCM) and of serum albumin (MTCBSA), hydrostatic pressure driven flux (JP), and osmotic filtration (JOSM) were calculated from the time-dependent volume and concentration data. All parameters have units of μL/min/cm2 and were compared to previously derived parameters from SD rats with a one-way ANOVA. There were no significant differences in the following (mean 6 SE): MTCM (CD1, 3.20 6 0.38, n = 7; C57bl, 2.34 6.41, n = 6; rat 2.72 6.25, n = 19; NS), JP (CD1, 0.42 6 0.19, n = 6; C57bl, 0.45 6 0.18, n = 6; rat 0.51 6 0.20, n = 9; NS), or JOSM (CD1, 0.92 6 0.35, n = 6; C57bl, 0.49 6 0.35, n = 6; rat 1.72 6 0.35, n = 6; NS). Only the MTCBSA demonstrated statistical difference: (x102: CD1, 9.52 6 2.34, n = 6; C57bl, 6.73 6 1.92, n = 7; rat 12.4 6 1.4, n = 17; p = .05). We conclude that use of a transport chamber to eliminate the variable peritoneal transfer area normalizes calculated transport characteristics and facilitates comparison between rodent species.