Hyaluronidase induces a transcapillary pressure gradient and improves the distribution and uptake of liposomal doxorubicin (Caelyx) in human osteosarcoma xenografts.

Liposomal drug delivery enhances the tumour selective localisation and may improve the uptake compared to free drug. However, the drug distribution within the tumour tissue may still be heterogeneous. Degradation of the extracellular matrix is assumed to improve the uptake and penetration of drugs. The effect of the ECM-degrading enzyme hyaluronidase on interstitial fluid pressure and microvascular pressure were measured in human osteosarcoma xenografts by the wick-in-needle and micropipette technique, respectively. The tumour uptake and distribution of liposomal doxorubicin were studied on tumour sections by confocal laser scanning microscopy. The drugs were injected i.v. 1 h after the hyaluronidase pretreatment. Intratumoral injection of hyaluronidase reduced interstitial fluid pressure in a nonlinear dose-dependent manner. Maximum interstitial fluid pressure reduction of approximately 50% was found after injection of 1500 U hyaluronidase. Neither intratumoral nor i.v. injection of hyaluronidase induced any changes in the microvascular pressure. Thus, hyaluronidase induced a transcapillary pressure gradient, resulting in a four-fold increase in the tumour uptake and improving the distribution of the liposomal doxorubicin. Hyaluronidase reduces a major barrier for drug delivery by inducing a transcapillary pressure gradient, and administration of hyaluronidase adjuvant with liposomal doxorubicin may thus improve the therapeutic outcome.

Interstitial fluid pressure and capillary diameter distribution in human melanoma xenografts.

Tumors have been shown to differ substantially in interstitial fluid pressure (IFP), but the biological properties of tumors governing the intertumor heterogeneity in IFP have not been identified conclusively. The purpose of the work reported here was to investigate whether the IFP of tumors is influenced significantly by the diameter distribution of the capillaries and hence by the geometric resistance of the capillary network to blood flow. Tumors of three human melanoma xenograft lines (D-12, R-18, U-25) showing similar capillary densities were included in the study. IFP was measured using the wick-in-needle technique. Capillary diameter distribution was determined by stereological analysis of histological sections. The lines differed significantly in tumor IFP (P < 0.05) and capillary diameter distribution (P < 0.05). Mean IFP was 6 mm Hg (D-12), 17 mm Hg (R-18), and 11 mm Hg (U-25). The mean of the mean capillary diameter was 13.1 microm (D-12), 10.9 microm (R-18), and 12.0 microm (U-25). The sequence of the lines from low to high IFP was the same as the sequence of the lines from large to small mean capillary diameter: D-12, U-25, R-18. Also, individual tumors of the same line differed substantially in IFP and in mean capillary diameter. IFP ranged from 2 to 15 mm Hg (D-12), from 2 to 36 mm Hg (R-18), and from 4 to 30 mm Hg (U-25). Mean capillary diameter ranged from 11.0 to 14.6 microm (D-12), from 9.5 to 11.7 microm (R-18), and from 10.4 to 13.0 microm (U-25). Inverse linear correlations between tumor IFP and mean capillary diameter were found for each of the melanoma lines [P < 0.05, R(2) = 0.85 (D-12); P < 0.05, R(2) = 0.86 (R-18); P < 0.01, R(2) = 0.93 (U-25)]. Moreover, the IFP and mean capillary diameter of individual tumors varied with tumor size in all lines. IFP decreased during tumor growth whereas mean capillary diameter increased with increasing tumor volume (P < 0.001). Taken together, these data suggest that the diameter distribution and hence the geometric resistance of the capillary network exerts significant influence on the IFP of tumors.

Measurement of proliferation activity in human melanoma xenografts by magnetic resonance imaging.

Tumor proliferation may be predictive for malignant progression and response to fractionated therapy of cancer. The purpose of the present work was to investigate whether the proliferation activity of solid tumors can be assessed in vivo from the proton relaxation times, T1 and T2. Tumors of four amelanotic human melanoma xenograft lines were studied. Three parameters were used to represent tumor proliferation activity; the volume doubling time, Tvol, the potential doubling time, Tpot, and the fraction of cells in S-phase. Tvol was determined from volumetric growth data. Tpot and S-phase fraction were determined by flow cytometric analysis of tumor cells after bromodeoxyuridine (BrdU) incorporation in vivo. T1 and T2 were measured by 1H-MRI in vivo, using spin-echo pulse sequences. The proliferation parameters and relaxation times differed considerably among the tumor lines. Significant correlations were found between the proliferation parameters and the relaxation times, regardless of whether Tvol, Tpot, or S-phase fraction was considered. Tumors with short Tvol and Tpot and high S-phase fraction had long T1 and T2 compared to tumors with long Tvol and Tpot and low S-phase fraction. The elongated T1 and T2 of fast growing tumors were probably due to increased interstitial and/or intravascular water content. The present results suggest that in vivo spin-echo 1H-MRI can be used to discriminate between tumors of high and low proliferation activity.

Interstitial fluid pressure, fraction of necrotic tumor tissue, and tumor cell density in human melanoma xenografts.

Interstitial fluid pressure (IFP) has been shown to differ substantially between individual tumors, but the tumor properties governing the intertumor heterogeneity in IFP have not been identified conclusively. The purpose of the work reported here was to investigate whether the fraction of necrotic tissue and the density of tumor cells are major determinants of the intertumor heterogeneity in IFP. The study was based on the hypothesis that the resistance against fluid flow in the tumor interstitium is influenced significantly by these parameters. Xenografted tumors of four human melanoma lines (A-07, D-12, R-18, U-25) were included in the study. Tumors showing large variation in necrotic fraction but similar cell densities (D-12, U-25) were used to study the influence of necrosis on IFP, whereas tumors showing no or insignificant necrosis but large variation in cell density (A-07, R-18) were used to search for correlations between IFP and cell density. IFP was recorded using the wick-in-needle technique. Necrotic fraction and cell density were measured by stereological analysis of histological sections using an image processing system. Significant correlations between IFP and necrotic fraction were not found, implying that the IFP of tumors is not influenced significantly by the development of necrosis. The R-18 tumors, which had a high cell density, showed a significantly higher IFP than the A-07 tumors, which had a low cell density. Significant correlations between IFP and cell density were not found when individual tumors of the same line were considered. These two observations suggest that the IFP of tumors depends on the cell density, but the cell density is probably not a major determinant of the IFP.

Vascular density in human melanoma xenografts: relationship to angiogenesis, perfusion and necrosis.

Studies of interrelationships between physiological parameters of tumours are sparse. The possibility that vascular density might be related to the rate of angiogenesis, the rate of perfusion and/or the development of necrosis was examined in the work reported here. Xenografted tumours of four human melanoma cell lines (A-07, D-12, R-18 and U-25) grown orthotopically in BALB/c nu/nu mice were included in the study. Vascular density and fraction of necrotic tissue were determined by stereological analysis of histological sections. The rate of angiogenesis was measured by using the intradermal angiogenesis assay. The rate of perfusion was studied by using the 86Rb uptake method. A-07 showed a higher vascular density, a higher rate of angiogenesis and a higher rate of perfusion than the other lines. D-12, R-18 and U-25, which differed significantly in the rate of angiogenesis, showed similar vascular densities and similar perfusion rates. Consequently, vascular density is not a sensitive measure of the rate of angiogenesis in the melanoma lines studied here, but might adequately reflect the perfusion rate. Significant necrotic regions developed in D-12 and U-25, but not in A-07 and R-18, presumably because large-diameter vessels, possibly arteriovenous shunts, occurred more frequently in D-12 and U-25 than in A-07 and R-18.

Proton relaxation times and interstitial fluid pressure in human melanoma xenografts.

The interstitial fluid pressure (IFP) and the proton spin-lattice and spin-spin relaxation times (T1 and T2) of some experimental tumours have been shown to be related to tumour water content. These observations have led to the hypothesis that magnetic resonance imaging (MRI) might be a clinically useful non-invasive method for assessment of tumour IFP. The purpose of the work reported here was to examine the general validity of this hypothesis. R-18 human melanoma xenografts grown intradermally in Balb/c nu/nu mice were used as the tumour model system. Median T1 and T2 were determined by spin-echo MRI using a 1.5-T clinical whole-body tomograph. IFP was measured using the wick-in-needle technique. No correlation was found between tumour IFP and fractional tumour water content. Moreover, there was no correlation between median T1 or T2 and IFP, suggesting that proton T1 and T2 values determined by MRI cannot be used clinically to assess tumour IFP and thereby to predict the uptake of macromolecular therapeutic agents.

Interstitial fluid pressure, perfusion rate and oxygen tension in human melanoma xenografts.

Interstitial fluid pressure (IFP) has been reported to be inversely correlated to rate of perfusion and oxygen tension (pO2) in experimental tumours (Lee et al., 1992; Roh et al., 1991 a). Studies of patients with squamous cell carcinoma of the uterine cervix have provided clinical data consistent with the experimental data (Roh et al., 1991b; Milosevic et al., 1995). These observations have led to the hypothesis that IFP might be a useful indicator of tumour oxygenation status. The purpose of the work reported here was to examine in detail the general validity of this hypothesis. R-18 human melanoma xenografts grown intradermally in Balb/c nu/nu mice were used as tumour model system. IFP and perfusion rate or IFP and pO2 were measured in the same individual tumours in two independent series of experiments. The wick-in-needle method was used to record IFP. Perfusion rate was studied by using the 86Rb uptake method. The KIMOC-6650 Eppendorf histograph was used to measure pO2. IFP, perfusion rate and pO2 differed considerably between individual tumours. However, there was no relationship between IFP and perfusion rate or IFP and pO2, suggesting that the oxygenation status of tumours cannot be derived from measurements of IFP. Consequently, IFP is probably not a useful predictor of radiation resistance caused by hypoxia.

Transient perfusion in human melanoma xenografts.

Studies of transplantable rodent tumours have suggested that malignant tissue might experience transient perfusion at the microvascular level. The purpose of the work reported here was to investigate whether transient perfusion can be demonstrated in xenografted human tumours. Tumours of four melanoma lines (A-07, D-12, R-18, U-25), grown orthotopically in Balb/c nu/nu mice, were included in the study. Transient perfusion was studied by using the double-fluorescent staining technique. Hoechst 33342 and DiOC7(3) were either administered simultaneously or Hoechst 33342 was administered 20 min before DiOC7(3). Detection of transient perfusion by this method requires that vessels are non-functional for at least 5 min owing to the distribution half-lives of the dyes in the blood. Usable combinations of dye concentrations were found by varying the concentrations of Hoechst 33342 and DiOC7(3) systematically. The level of perfusion mismatch following simultaneous administration of the dyes ranged from approximately 1.5% for U-25 tumours to approximately 3.0% for R-18 tumours at these combinations. Moreover, the fraction of vessels stained only with Hoechst 33342 and the fraction of vessels stained only with DiOC7(3) were not significantly different whether the dyes were administered simultaneously or sequentially. Transient perfusion could not be demonstrated in any of the tumour lines. Thus, the fraction of vessels stained only with Hoechst 33342 and the fraction of vessels stained only with DiOC7(3) were not significantly higher after sequential than after simultaneous administration of the dyes. Moreover, the vessels stained only with Hoechst 33342 and the vessels stained only with DiOC7(3) were randomly distributed within the tumours whether the dyes were administered simultaneously or sequentially. Consequently, acute hypoxia caused by transient perfusion is probably a less pronounced phenomenon in malignant tissue than previous studies of rodent tumours have suggested.

Interstitial fluid pressure in human melanoma xenografts. Relationship to fractional tumor water content, tumor size, and tumor volume-doubling time.

The interstitial fluid pressure (IFP) has been shown to be elevated in malignant tissue, but the possibility that IFP might be related to other pathophysiological parameters of the tissue has not been fully explored. The purpose of the study here reported was to measure the IFP in human melanoma xenografts and to search for possible correlations between tumor IFP and fractional tumor water content, tumor wet weight, or tumor volume-doubling time. Tumors of four melanoma lines (A-07, D-12, R-18, U-25), grown orthotopically in BALB/c-nu/nu mice, were included in the study. Tumor IFP, measured by using the wick-in-needle technique, ranged from 2 to 10 mm Hg (D-12), from 2 to 15 mm Hg (A-07 and U-25), and from 2 to 30 mm Hg (R-18). Statistically significant correlations between tumor IFP on the one hand and fractional tumor water content, tumor wet weight, or tumor volume-doubling time on the other were not found, whether the tumor lines were analyzed individually or together. These observations suggest that simple general relationships between the IFP and the other pathophysiological parameters measured here, might not exist in tumors.