A pilot study of the effects of mild systemic heating on human head and neck tumour xenografts: Analysis of tumour perfusion, interstitial fluid pressure, hypoxia and efficacy of radiation therapy

Abstract

Purpose: The tumour microenvironment is frequently hypoxic, poorly perfused, and exhibits abnormally high interstitial fluid pressure. These factors can significantly reduce efficacy of chemo and radiation therapies. The present study aims to determine whether mild systemic heating alters these parameters and improves response to radiation in human head and neck tumour xenografts in SCID mice.

Materials and methods: SCID mice were injected with FaDu cells (a human head and neck carcinoma cell line), or implanted with a resected patient head and neck squamous cell carcinoma grown as a xenograft, followed by mild systemic heating. Body temperature during heating was maintained at 39.5 ± 0.5 °C for 4 h. Interstitial fluid pressure (IFP), hypoxia and relative tumour perfusion in the tumours were measured at 2 and 24 h post-heating. Tumour vessel perfusion was measured 24 h post-heating, coinciding with the first dose of fractionated radiotherapy.

Results: Heating tumour-bearing mice resulted in significant decrease in intratumoural IFP, increased the number of perfused tumour blood vessels as well as relative tumour perfusion in both tumour models. Intratumoural hypoxia was also reduced in tumours of mice that received heat treatment. Mice bearing FaDu tumours heated 24 h prior to five daily radiation treatments exhibited significantly enhanced tumour response compared to tumours in control mice.

Conclusions: Mild systemic heating can significantly alter the tumour microenvironment of human head and neck tumour xenograft models, decreasing IFP and hypoxia while increasing microvascular perfusion. Collectively, these effects could be responsible for the improved response to radiotherapy.

Keywords: Human head and neck cancer; hypoxia; interstitial fluid pressure; radiation therapy; tumour perfusion.

Figure 1

Interstitial fluid pressure decreases and perfusion measured by LDF increases following systemic heating (n = 5). (A) Plot of IFP measured in FaDu tumours 2 h and 24 h after systemic heating to 39.5 °C for 4 h. A significant reduction in IFP was observed in the tumours in heated mice as compared to those in unheated control animals (ANOVA p <0.01**). (B) Plot of LDF measurements on FaDu in blood perfusion units (BPU) in tumours showing significant increase in relative tumour perfusion in heated mice at 2 h and 24 h post-heating in comparison to those in unheated animals (ANOVA p <0.001***). (C) Plot of tumour interstitial fluid pressures (IFP) in xenografts of a resected patient head and neck tumour (no. 19705) grown subcutaneously in SCID mice at 2 h and 24 h after systemic heating to 39.5 °C for 4 h. IFPs in tumours post-heating show significant decrease (ANOVA p <0.01**). (D) Plot of relative tumour perfusion measurements using LDF on mice bearing the same patient tumour xenografts show small increases in relative tumour perfusion in heated mice at both 2 h and 24 h post-heating. The increases were not statistically significant.

Figure 2

Tumour vascular perfusion is increased following systemic heating of mice bearing FaDu tumours (n = 2). Fluorescent liposomes were injected 2 h post-heating when body temperature has returned to homeostatic level. (A) Representative fluorescence micrographs (10×) showing perfusion of FaDu tumour vessel in unheated control and in heated mice, and (B) the corresponding binary images. (C) Plot of the number of perfused vessels in the tumours determined using NIH ImageJ particle analysis routine shows an increase in the number of perfused vessels in tumour from mice that were heated (average 92 in two tumours) and unheated control (average 33 in two tumours). Scale bar = 100 μm.

Figure 3

The number of perfused vessels in tumours is increased following the heating of patient tumour xenograft tumour-bearing mice (n = 3) after systemic heat treatment. (A) Representative fluorescence micrographs (20×) showing perfused blood vessels in patient tumour (no. 19705) xenografts in unheated control (left) and in heated mice (right). Circles highlight representative vessels; the brightest intensity is the vessel and the grey is extravasated liposomes. (B) Plot of the number of perfused vessels determined using NIH ImageJ on the micrographs above. The numbers of perfused vessels show statistically significant increase. Scale bar = 100 μm (Student t-test *p <0.05).

Figure 4

Mild systemic heating improves tumour oxygenation. (A) Hypoxic areas in tumour sections were identified using immunohistochemistry (IHC) to label pimonidazole hydrochloride (Hypoxyprobe-1) adducts of proteins and peptides that are formed at low oxygen tension. Panels on the left are micrographs (10×) from tumour sections from a control unheated mouse and from heated mice 2 h and 24 h post-heating. Panels on the right are binary images of the micrographs on the left; the binary threshold was done using NIH ImageJ. The micrographs show extensive hypoxic areas in the control tumour and in the tumour 2 h post-heating. The hypoxic area is much reduced at 24 h post-heating. (B) A plot of the number of IHC stained sections from unheated control mice and those from mice at 2 h and 24 h post-heating. The pixel counts were obtained using NIH ImageJ. There was no significant decrease in the number of hypoxic pixels at 2 h post-heating but at 24 h post-heating there was significant decrease in hypoxia in the tumours. Scale bar = 100 μm (n = 3 per group, ANOVA *p <0.05; **p <0.01).

Figure 5

Radiation efficacy is improved by heat treatment prior to radiation therapy. Plot of growth of FaDu tumour xenografts in four groups (n = 5). Group 1 unheated control, group 2 heated (day 24), group 3 local radiation at 2Gy on days 25–29, group 4 heating on day 24 followed by 2Gy local radiation on days 25–29 starting 24 h post-heating. Tumour growth in only group 4, heating and radiation showed significant tumour response to therapy as compared to the control group (ANOVA **p <0.01).