Preirradiated Tissue Research Articles

The Preparation of the Recipient Site in Fat Grafting: A Comprehensive Review of the Preclinical Evidence.

Several methods to prepare the recipient site in fat grafting have been proposed in recent decades. However, to date, these procedures have never been reviewed exhaustively. The purpose of the present study is to provide a comprehensive overview of the different techniques to prepare the recipient site for fat grafting as they were investigated in preclinical studies, with resulting outcomes and underlying mechanisms of action. The PubMed/MEDLINE database was queried to search for preclinical investigations on the preparation of the recipient site in fat grafting using the following algorithm: ((recipient site) AND (fat grafting) OR (lipofilling) OR (lipograft)). A priori criteria were applied to review the resulting articles. Thirteen animal studies met inclusion criteria. Overall, five techniques were identified: external volume expansion, implantation of alloplastic material (silicone sheets), administration of cell-proliferation factors (i.e., vascular endothelial growth factor, adipose tissue-derived stromal vascular fraction, and interleukin-8), ischemia, and microneedling. A positive effect on cellular activity (cell proliferation and angiogenesis) was demonstrated by all studies and achieved with all techniques. Seven of the eight authors who examined this aspect reported enhancement of fat graft survival. Improvement of fat grafting surgical outcomes is documented preclinically using different recipient-site preparation techniques, particularly through enhancement of vascularization and soft-tissue expansion. This understanding will lead to further clinical research, especially for those cases where improvement of the recipient site is recommended, such as contracted scars or preirradiated tissues.

In-vivo quantification of the revascularization of a human acellular dermis seeded with EPCs and MSCs in co-culture with fibroblasts and pericytes in the dorsal chamber model in pre-irradiated tissue.

Transplantation of a cell-seeded graft may improve wound healing after radiotherapy. However, the survival of the seeded cells depends on a rapid vascularization of the graft. Co-culturing of adult stem cells may be a promising strategy to accelerate the vessel formation inside the graft. Thus, we compared the in vivo angiogenic potency of mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) using dorsal skinfold chambers and intravital microscopy. Cells were isolated from rat bone marrow and adipose tissue and characterized by immunostaining and flow cytometry. Forty-eight rats received a dorsal skinfold chamber and were divided into 2 main groups, irradiated and non-irradiated. Each of these 2 groups were further subdivided into 4 groups: unseeded matrices, matrices+fibroblasts+pericytes, matrices+fibroblasts+pericytes+MSCs and matrices+fibroblasts+pericytes+EPCs. Vessel densities were quantified semi-automatically using FIJI. Fibroblasts+pericytes-seeded matrices showed a significantly higher vascular density in all groups with an exception of non-irradiated rats at day 12 compared to unseeded matrices. Co-seeding of MSCs increased vessel densities in both, irradiated and non-irradiated groups. Co-seeding with EPCs did not result in an increase of vascularization in none of the groups. We demonstrated that the pre-radiation treatment led to a significant decreased vascularization of the implanted grafts. The augmentation of the matrices with fibroblasts and pericytes in co-culture increased the vascularization compared to the non-seeded matrices. A further significant enhancement of vessel ingrowth into the matrices could be achieved by the co-seeding with MSCs in both, irradiated and non-irradiated groups.

Letter to the Editor concerning “Gene expression analysis in chronic postradiation proctopathy” by Traub et al. (Int J Colorectal Dis 27:879–884, 2012)

Dear Editor: With interest, we read the paper by Traub et al. providing important information on the molecular mechanism of radiation-induced proctitis. Nevertheless, we would like to use the opportunity to address a word of caution from a more clinical point of view according to the potential danger of biopsies in the irradiated rectum. Biopsies of the rectal wall in preirradiated tissue might cause devastating complications. For example, Shakespeare et al. [1] described six patients with a recto-urethral fistula after brachytherapy for prostate cancer. Three of these patients developed the fistula 3–4 months after transrectal biopsy. Performing invasive procedures in radiated and severely contaminated tissue may cause severe infectious complications as well as nonor slow-healing ulcerating wounds [2]. Theodorescu et al. [3] also pointed out the risk of severe complications after invasive procedures such as biopsies. Traub et al. reported that patients were selected based on the following parameters: (1) after radiotherapy for cervical or prostate cancer, (2) LENT/RTOG grade 3/4, and (3) acute rectal bleeding with the need of endoscopic evaluation/treatment. Biopsies were taken from the affected rectum (ventral and dorsal part). For patients having a history of radiotherapy in the pelvic region, we would recommend that invasive procedures, such as biopsies in the anorectal region, should be avoided because of the above-mentioned complications. If a biopsy is needed to exclude malignancy, in our opinion, this should be done only after all noninvasive procedures have been done, and these could not rule out malignancy. Others also have these recommendations [4]. Thus, we were quite amazed that biopsies were taken for research purposes. Patients need to be aware prior to endoscopy that even small biopsy lesions might lead to long-lasting complications and consecutive treatment. In this context, biopsies for research on postradiation proctopathy remain questionable.

Abstract 1540: Inhibition of SDF production by brain tumor cells reduces brain tumor bed effect

Abstract Previous study on a murine prostate tumor model, TRAMP-C1, has shown that tumor transplants in preirradiated (pre-IR) tissues have longer growth delay and lower microvascular density (MVD) than in nonirradiated tissues, a phenomenon known as the tumor bed effect (TBE) that is associated with less responsive to salvage radiotherapy or chemotherapy and higher risk of metastasis. This study further demonstrates that a murine astrocytoma, ALTS1C1, grown from pre-IR brain tissues also have obvious TBE. This indicates that there is also a brain TBE that may affect the therapeutic efficacy of recurrent brain tumor after radiotherapy. Additional study shows that the inhibition of SDF expression in ALTS1C1 by specific siRNA reduces pre-IR-induced tumor growth delay and TAM association with hypoxia. The vessel formation mechanism is also altered into more mature form. This indicates that SDF production by brain tumor cells plays significant roles on the response of recurrent brain tumors after radiotherapy to salvage therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1540. doi:1538-7445.AM2012-1540

Vasculatures in Tumors Growing From Preirradiated Tissues: Formed by Vasculogenesis and Resistant to Radiation and Antiangiogenic Therapy

To investigate vasculatures and microenvironment in tumors growing from preirradiated tissues (pre-IR tumors) and study the vascular responses of pre-IR tumors to radiation and antiangiogenic therapy. Transgenic adenocarcinoma of the mouse prostate C1 tumors were implanted into unirradiated or preirradiated tissues and examined for vascularity, hypoxia, and tumor-associated macrophage (TAM) infiltrates by immunohistochemistry. The origin of tumor endothelial cells was studied by green fluorescent protein-tagged bone marrow (GFP-BM) transplantation. The response of tumor endothelial cells to radiation and antiangiogenic agent was evaluated by apoptotic assay. The pre-IR tumors had obvious tumor bed effects (TBE), with slower growth rate, lower microvascular density (MVD), and more necrotic and hypoxic fraction compared with control tumors. The vessels were dilated, tightly adhered with pericytes, and incorporated with transplanted GFP-BM cells. In addition, hypoxic regions became aggregated with TAM. As pre-IR tumors developed, the TBE was overcome at the tumor edge where the MVD increased, TAM did not aggregate, and the GFP-BM cells did not incorporate into the vessels. The vessels at tumor edge were more sensitive to the following ionizing radiation and antiangiogenic agent than those in the central low MVD regions. This study demonstrates that vasculatures in regions with TBE are mainly formed by vasculogenesis and resistant to radiation and antiangiogenic therapy. Tumor bed effects could be overcome at the edge of larger tumors, but where vasculatures are formed by angiogenesis and sensitive to both treatments. Vasculatures formed by vasculogenesis should be the crucial target for the treatment of recurrent tumors after radiotherapy.

Enhanced response to C225 of A431 tumor xenografts growing in irradiated tumor bed

Background and purpose We recently demonstrated that C225 maintenance therapy after completion of radiotherapy further increased tumor radiocurability. The present study assessed mechanisms underlying the observed improvement in C225 efficacy in pre-irradiated tissue (tumor bed). Materials and methods A431 xenografts growing in pre-irradiated and non-irradiated tissue were treated with C225. Tumors were assessed for growth delay, cell proliferation, hypoxia, EGFR and VEGF expressions. In vitro clonogenic survival of cells derived from these tumors was also assayed. Results Pre-irradiation of tumor bed induced growth retardation, reduction in Ki-67 labeling, and overexpression of HIF-1α, CA IX, EGFR and VEGF biomarkers. C225 treatment dramatically inhibited tumor growth in the irradiated tumor bed ( P < 0.0001), which was associated with further reduction in Ki-67 labeling, and reduced expression of HIF-1α, CA IX, EGFR and VEGF. Cells derived from tumors in the pre-irradiated bed showed increased sensitivity to C225. C225 was more cytotoxic against hypoxic than well-oxygenated A431 cells grown in vitro. Conclusion A431 xenografts growing in pre-irradiated tumor bed exhibit enhanced sensitivity to C225. Pre-irradiated tissue microenvironment seems to render tumor cells more susceptible to C225 cytostatic and cytotoxic actions. If confirmed in other tumor models these findings support the use of C225 maintenance therapy after completion of radiotherapy.

Mechanisms of resistance to ionizing radiation in rectal cancer

While patients with breast cancers are not subjected to the adverse side effects of tamoxifen or trastuzumab if their tumors are negative for estrogen, progesterone or Her-2/Neu, neoadjuvant ionizing radiation with concurrent chemotherapeutic agents is administered almost universally to patients with stage II/III rectal cancers. There is, however, a tremendously wide range of response to this preoperative modality from complete pathological response to continuous tumor growth in patients receiving the same form of treatment. The specific phenotype of the tumor plays a major role in rendering tumor cells survival advantage to the cytotoxic effects of chemoradiation. Pathways such as proliferation, cell cycle, apoptosis and hypoxia have been investigated under a variety of conditions in preirradiated tissues and postirradiated tumors. This article reviews the current evidence available to identify a molecular profile predictive of the best response to ionizing radiation.

Reirradiation of recurrent breast cancer with and without concurrent chemotherapy

BackgroundTreatment options for loco-regional recurrent breast cancer after previous irradiation are limited. The efficacy of chemotherapy might be hampered because of impaired tissue perfusion in preirradiated tissue. Thus, mastectomy or local excision and reconstructive surgery are the preferred treatments. However, in recent years evidence accumulates that a second breast conserving approach with reirradiation as part of the treatment might be feasible and safe and, furthermore, reirradiation might be an option for palliation. Here we report on the experience of a single community centre in reirradiation of recurrent breast cancer.MethodsThe report is based on 29 patients treated with reirradiation. All data were prospectively collected. The median age was 63 years (range 35 to 82 yrs). The interval between initial diagnosis and diagnosis before start of reirradiation was 11.6 months to 295.5 months. The mean total dose (initial dose and reirradiation dose) was 106.2 Gy (range 80.4 to 126 Gy) and the mean BED3 Gy 168,5 Gy (range 130,6 to 201,6). The mean interval between initial radiotherapy and reirradiation was 92.9 months (range 8.7 to 290.1). Inoperable or incompletely resected patients were offered concurrent chemotherapy with either 5-FU or capecitabine. All patients received 3D-conformal radiotherapy with 1.6 to 2.5 Gy/fraction five times weekly. The treatment volume comprised all visible lesions or lesions detectable on CT/MRI/FDG-PET/CT or the tumour bed or recurrent tumour.ResultsThe local progression-free survival of all patients at one and two years was 81% and 63%. Patients who had no surgery of the recurrence (16/29) had local progression-free survival at one and two years of 72% and 25% with a median progression-free survival time of 17 months. Partial remission and good symptom relief was achieved in 56% (9/16) or complete response of symptoms and/or tumour in 44% (7/16). Patients who had no distant metastases and had at least an R1-resection had a local progression-free survival of 90% after 2 years. The disease-free survival after 2 years was 43% and the median disease-free survival time was 24 months. In four patients a second breast conserving operation was performed and the cosmetic results in all four patients are good to excellent. Acute side effects were mild to moderate with no grade 3 or 4 toxicity. Accordingly, no grade 3 or 4 late effects were observed so far. No grade 3 or 4 plexopathy was observed.ConclusionIn this heterogeneous group of patients reirradiation of locoregional recurrences of breast cancer showed low to moderate acute toxicity. In our experience, local control rates are high and palliation is good.

CYR61 and αVβ5 Integrin Cooperate to Promote Invasion and Metastasis of Tumors Growing in Preirradiated Stroma

Radiotherapy is widely used to treat human cancer. Patients locally recurring after radiotherapy, however, have increased risk of metastatic progression and poor prognosis. The clinical management of postradiation recurrences remains an unresolved issue. Tumors growing in preirradiated tissues have an increased fraction of hypoxic cells and are more metastatic, a condition known as tumor bed effect. The transcription factor hypoxia inducible factor (HIF)-1 promotes invasion and metastasis of hypoxic tumors, but its role in the tumor bed effect has not been reported. Here, we show that tumor cells derived from SCCVII and HCT116 tumors growing in a preirradiated bed, or selected in vitro through repeated cycles of severe hypoxia, retain invasive and metastatic capacities when returned to normoxia. HIF activity, although facilitating metastatic spreading of tumors growing in a preirradiated bed, is not essential. Through gene expression profiling and gain- and loss-of-function experiments, we identified the matricellular protein CYR61 and alphaVbeta5 integrin as proteins cooperating to mediate these effects. The anti-alphaV integrin monoclonal antibody 17E6 and the small molecular alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 suppressed invasion and metastasis induced by CYR61 and attenuated metastasis of tumors growing within a preirradiated field. These results represent a conceptual advance to the understanding of the tumor bed effect and identify CYR61 and alphaVbeta5 integrin as proteins that cooperate to mediate metastasis. They also identify alphaV integrin inhibition as a potential therapeutic approach for preventing metastasis in patients at risk for postradiation recurrences.

Transforming growth factor beta 1 dependent regulation of Tenascin-C in radiation impaired wound healing

Background Following preoperative radiotherapy prior to ablative surgery of squamous epithelial cell carcinomas of the head and neck region fibrocontractive wound healing disorders occur. Tenascin-C is significantly increased in fibrotic tissue conditions and can be stimulated by the transcription factor NFκB p65. Previous studies showed a reduction of irradiation induced fibrosis during the wound healing process by anti-TGFβ 1-treatment. The aim of the study was to clarify the question whether Tenascin-C expression is elevated in radiation impaired wounds and whether anti-TGFβ 1-treatment is capable to influence Tenascin-C and NFκB expression. Material and methods Wistar rats (male, weight 300–500 g) underwent preoperative irradiation of the head and neck region with 40 Gy, fractionated four times 10 Gy (16 animals), whereas 8 non-irradated animals served as a control. Four weeks after irradiation a free myocutaneous gracilis flap taken from the groin was transplanted to the neck. Eight animals additionally received 5 μg anti-TGFβ 1 into the graft bed by intradermal injection prior to each fraction of irradiation and on days 1–7 post-operation. On day 14 and 28 following surgery immunohistochemistry (ABC–POX method) was performed assessing the cytoplasmic NFκB and Tenascin-C staining in the transition area between transplant and graft bed. For quantitative considerations the labeling index (ratio: positive cells/total cells) was determined. Results A significantly altered expression of Tenascin-C in the preirradiated tissue was observed following anti-TGFβ 1-treatment. NFκB protein was upregulated in irradiated animals and was significantly reduced in the anti-TGFβ 1 treated group on day 28 after transplantation. Conclusions Tenascin-C expression is prolonged in irradiated animals as compared to non-irradiated tissue. Tenascin-C seems to be regulated by TGFβ 1 as the application of TGFβ 1-neutralizing antibodies reduces Tenascin-C expression. Tenascin-C is a potentially useful marker for tissue remodeling due to its restricted distribution in adult and healthy tissue and a hallmark for developing fibrosis.

Smad-3 and Smad-7 expression following anti-transforming growth factor beta 1 (TGFβ 1)-treatment in irradiated rat tissue

Background and purpose Wound healing disorders and the development of fibrosis following surgery in preirradiated tissue are clinically well characterised and may even become more pronounced with increasing use of neoadjuvant radiochemotherapy. The cytokine transforming growth factor beta 1 TGFβ 1 has a key role either in the wound healing process or induction of fibrosis. Following activation of the TGFβ receptors, intracellular signal transduction is mediated by a variety of Smad proteins. Smad-3 acts as an activator of signal transduction, whereas Smad-7 has an inhibitory effect. This study evaluated the biological effect of neutralizing TGFβ 1 antibodies, the relationship between TGFβ 1 and Smad-3/7 expression and changes of collagen synthesis, following inhibition of TGFβ 1 activity in irradiated rat tissue. Patients and methods A total of 45 Wistar rats (male, weight 300–450 g) were used. A free myocutaneous gracilis flap was transplanted in all rats and animals were allocated into 3 groups as follows: Group 1 ( n=15 rats) treated with surgery alone (TX); Group 2 ( n=15 rats) preoperative radiotherapy (RT) followed by TX; Group 3 ( n=15 rats) RT+TX+anti-TGFβ 1-treatment. The interval between end of radiotherapy and grafting was 10 days. For anti-TGFβ 1 treatment, 1 μg anti-TGFβ 1/500 μl PBS were locally applied s.c. intraoperatively and on day 1 to 7 after surgery. Tissue samples were collected perioperatively and on days 3, 7, 14, 28 following surgery from the transition area between the graft and the graft bed. ECM synthesis and expression of TGFβ 1, Smad-3, Smad-7, prolyl-hydroxyprolinase-β were quantitated by immunohistochemistry (labelling indices). Changes in collagen synthesis were assessed qualitatively by polarisation microscopy of sirius red staining and collagen I immunhistochemistry. The different regulation of inhibitory Smad-7 was detected in irradiated and irradiated plus anti-TGFβ 1 treated animals by semiquantitative RT-PCR and the nucleocytoplasmic shuttling of phosphorylated Smad-3 was shown by isolation of nuclear proteins and Western blot analysis. Results Following anti-TGFβ 1 treatment, an attenuated expression of TGFβ 1, a reduction in EMC synthesis and fibrosis could be observed in irradiated tissue compared to the irradiated tissue without anti-TGFβ 1-treatment. While preoperative RT increases the expression of Smad-3/7 proteins, an upregulation of Smad-7 from day 3 until day 14 following surgery and a downregulation of Smad-3 on day 14 after surgery could be observed following anti-TGFβ 1-treatment. The samples which were irradiated alone displayed a reduced signal for Smad-7 mRNA and an induction of Smad-3 protein phosphorylation shown by nucleocytoplasmic shuttling. In contrast, the anti-TGFβ 1-treated samples showed an increase in Smad-7 mRNA and a downregulation of Smad-3 phosphorylation. Prolyl-hydroxyprolinase-β expression and collagen I synthesis were reduced following anti-TGFβ 1-treatment. Conclusions A reduction of Smad-3 proteins in parallel with an increase of Smad-7 may contribute to the inhibitory effect and the reduction of ECM synthesis after blocking of TGFβ 1 activity by treatment with neutralizing antibodies. This may indicate a molecular mechanism in the therapeutic intervention to reduce fibrosis, hypertrophic scar formation and chronic wound healing disorders.

Improved free vascular graft survival in an irradiated surgical site following topical application of rVEGF

Purpose Wound healing disorders following surgery in preirradiated tissue are clinically well known and may even become more crucial with the increasing use of neoadjuvant chemoradiation protocols. Both the expression of vascular endothelial growth factor (VEGF) and endoglin (CD105) play a key role in neovascularization and wound healing after soft tissue grafts in irradiated and nonirradiated tissue. Modulation of neovascularization through the application of recombinant VEGF (rVEGF) may be a therapeutic option to reduce wound healing disorders in irradiated tissue. An experimental in vivo model was used to study the possible role of rVEGF for reduction of wound healing disorders and the promotion of neovascularization. Methods and materials A free myocutaneous gracilis flap was transplanted from the groin into the neck region of Wistar rats (weight 300–500 g) with and without previous irradiation of the neck region with 40 Gy: Group 1 ( n = 7) radiotherapy alone; Group 2 ( n = 14) flap transplantation alone and rVEGF; Group 3 ( n = 14) radiotherapy, transplantation, and rVEGF. Time interval between irradiation and grafting was 10 ± 1 day. 1.0 μg rVEGF/500 μL phosphate-buffered saline was applied s.c. intraoperatively and on Days 1 through 7. Neovascularization (CD105) and endogenous VEGF expression were analyzed by means of immunohistochemistry on Days 3, 5, 7, 14, and 28 postoperatively and quantified as labeling indices (LI). Results After irradiation there was a continuous significant reduction of the cytoplasmic VEGF expression (mean LI: 0.018 ± 0.048) compared with the nonirradiated control (mean LI: 0.042 ± 0.006) ( p < 0.001). VEGF expression after flap transplantation without irradiation after VEGF application was at a constantly higher level from Day 3 (mean LI: 0.044 ± 0.01) to Day 28 postoperatively compared with the control group (Day 3, mean LI: 0.028 ± 0.006) ( p < 0.001). As an indication of increased neovascularization after the local application of rVEGF, a significantly increased expression of CD105 was found in the transition area and graft bed from Day 7 on ( p < 0.001). After irradiation and grafting there was a significant overall increase in the VEGF- and CD105-expression throughout Day 28 after rVEGF in the transition area ( p < 0.001). Conclusions Whereas irradiation alone led to a downregulation of the endogenous VEGF expression, rVEGF application resulted in an increased expression and in a CD105 associated neovascularization after soft tissue grafting in irradiated tissues. Application of rVEGF may enable modulation of wound healing by influencing neovascularization. This could indicate a possible clinical approach for reducing fibrosis and chronic wound healing disorders in irradiated tissues.

Transforming growth factor-beta receptor-II up-regulation during wound healing in previously irradiated graft beds in vivo.

Wound healing disorders may often present in patients with head and neck cancer after surgical interventions, particularly in preirradiated tissue. Inflammatory changes and the expression of cytokines can lead to induction of fibrosis. The isoforms of the transforming growth factor beta (TGFbeta1-3) play a key role for this process. It has been shown that radiation treatment associated fibrosis is induced by TGFbeta1 and TGFbeta2, although the influence of radiation on the expression of the TGFbeta receptor-II (TGFbetaR-II) involved in the signal transduction of TGFbeta remains elusive. The objective of this in vivo study was to analyze the expression profile of TGFbetaR-II in the graft bed and in the transition area between graft and graft bed after surgery with and without prior radiation treatment to compare with the expression profiles of activated TGFbeta1 and latency-associated peptide. A total of 48 Wistar rats (male, weight 300-500 g) were used in the study. Eighteen rats were irradiated in the neck region (3 x 10 Gy) without transplantation. A free myocutaneous gracilis flap was transplanted in 30 rats, of which 16 animals were preirradiated in the neck region (3 x 10 Gy) and 14 animals were not irradiated at all. Tissue samples were taken postoperatively from the transition area between the graft and the graft bed and from the graft bed itself after 3, 7, 14, and 28 days. Tissue samples were taken from the irradiated neck region and the non-irradiated groin region 0, 4, 7, 11, 14, and 28 days after the end of the exposure. The expression of TGFbetaR-II, activated TGFbeta1 and latency-associated peptide was analyzed immunohistochemically both qualitatively and quantitatively (labeling index). The success rate for graft healing was 75% in the previously irradiated group with 30 Gy, and 86% in the non-irradiated group. Following radiation alone a significantly (p = 0.04) increased TGFbetaR-II expression in the neck was revealed 2-4 weeks following irradiation compared to non-irradiated skin. Whereas only minor differences in TGFbetaR-II expression were observed following surgery between the groups with and without prior radiation in the transition area between the graft and the graft bed, the group undergoing prior radiation and subsequent grafting showed significantly increased expression in the bed compared to the non-preirradiated group with a maximum on postoperative day 7 (week 1, p = 0.003; week 2-4, p < 0.001). In irradiated tissues the up-regulation of TGFbetaR-II expression correlated with an increase of activated TGFbeta1 and latency-associated peptide expression compared to non-irradiated tissues. After irradiation, a significantly increased TGFbetaR-II expression was identified in the irradiated graft bed, which may be the reason for delayed reepithelialization and fibrosis. Exogenous blocking or TGFbetaR-II inhibitors could therefore represent a new therapeutic approach for improving wound healing after preoperative radiotherapy.

Impact of the tumour bed effect on microenvironment, radiobiological hypoxia and the outcome of fractionated radiotherapy of human FaDu squamous-cell carcinoma growing in the nude mouse

Purpose : To investigate the impact of the tumour bed effect (TBE) on histological parameters of the micromilieu, radiobiological hypoxic fraction and local control after fractionated irradiation in FaDu squamous-cell carcinoma in the nude mouse. This tumour has previously shown a clear-cut TBE caused by increased necrotic cell loss at a constant cell production rate in the viable tumour compartment. Materials and methods : Human FaDu tumours were studied in the NMRI nude mouse. Tumours were transplanted either into unirradiated subcutaneous (s.c.) tissues (controls) or s.c. tissues pre-irradiated with 12.5 Gy (TBE group). In both groups we measured the volume doubling time (VDT), potential doubling time (T pot) , relative necrotic area, and in the viable tumour compartment the relative vascular area (9F1 mAb), relative hypoxic area (NITP or pimonidazole), relative perfused area (Hoechst 33342), and the perfused fraction of vasculature. The tumour control dose 50% (TCD 50), radiobiological hypoxic fraction (rHF) and dose-modifying factors (DMF) for the comparison of tumours in the TBE and control groups were determined from local tumour control data after treatment with single doses under ambient conditions or under clamp hypoxia, and after irradiation with 30 fractions under ambient conditions within 6 weeks using maximum-likelihood analysis. Results : A clear-cut TBE (VDT = 4.0 days (95%CI 2.9;4.4) for the control group versus 7.2 days (6.4;8.9) for the TBE group; p <0.0001) caused by increased necrosis (mean relative necrotic area of 12% (5;20)) versus 33% (10;41); p = 0.07) at a constant cell production rate (T pot = 2.2 days (1.4;2.3) versus 2.2 days (1.7;2.6); p = 0.30) was confirmed. Histological analysis of the micromilieu within the vital subarea revealed no systematic differences between the TBE and control groups. The rHF of 2% (0.1;27) for control tumours was lower than the 15% (95% CI 2;91) for the TBE group, but this difference was nonsignificant (p = 0.12). Compared with control tumours, the TCD 50 for irradiation under clamped hypoxia was in a statistical trend lower for tumours in the TBE group (DMF 1.11 (0.98;1.28), p = 0.09). After fractionated irradiation, tumours of the TBE group were significantly more radiosensitive (TCD 50 56.6 Gy (46;70) versus 78.7 Gy (63;100); p = 0.003). Conclusions : The results on FaDu tumours growing in pre-irradiated tissues indicate that increased necrosis caused by impairment of the vascular supply may increase the radiosensitivity of tumours treated by fractioned irradiation.

Impact of Tumor Stroma on Expression of the Tumor Bed Effect in R1H Rat Rhabdomyosarcoma

The impact of intratumoral stroma on the expression of the tumor bed effect was studied in R1H rat rhabdomyosarcoma. For this, either R1H tumors growing in subcutaneous tissues (in situ) were irradiated at median volumes of 40 mm3 with a single exposure of 15 Gy (200 kVp X rays), or fresh tumor fragments or cloned tumor cells cultured in vitro were transplanted into subcutaneous tissue 2 days after preirradiation with 15 Gy. The tumor bed effect was evaluated by the time necessary for experimental tumors to grow from 0.1 cm3 to 10 cm3. When irradiated in situ, R1H tumors expressed a clear-cut tumor bed effect. The growth rate was decreased by a factor of 1.8 +/- 0.2 compared to controls. A significant decrease in growth rate by a factor of 1.7 +/- 0.2 was also observed when cultured tumor cells were transplanted into preirradiated tissue. However, when tumor fragments were transplanted into preirradiated sites, no significant decrease in growth rate occurred in two independent experiments (1.0 +/- 0.1; 1.2 +/- 0.2). Our results indicate that vessels originating from the intratumoral stroma can modulate the expression of the tumor bed effect in R1H tumors.

The Influence of Irradiation of the Tumor Bed on Tumor Hypoxia: Measurements by Radiation Response, Oxygen Electrodes, and Nitroimidazole Binding

SCCVII carcinomas and RIF-1 sarcomas were transplanted and grown in the unirradiated (control tumors) or preirradiated tissue of mice. Tumor oxygenation was measured when the tumors were in the size range 50-150 mm3 using three different methods. The tumors growing in tissues preirradiated with 20 Gy were significantly more hypoxic than the control tumors for both tumor types. Radiobiologically hypoxic fractions of these tumors were approximately 10 times higher than those of the controls. The relative [3H]misonidazole binding of the tumors transplanted into irradiated tissue was approximately five times (P < 0.01) higher in SCCVII tumors or approximately two times (P < 0.05) higher in RIF-1 tumors than that of the controls. The pO2 histographs measured directly by a polarographic microelectrode were more hypoxic (P < 0.001) and changed from bimodal to unimodal in pattern in the tumors transplanted into irradiated tissue compared to those of the controls. The increase of the relative [3H]misonidazole binding in the tumors transplanted into irradiated tissue compared to that of the controls was similar to the increase of the cumulative frequency of pO2 below 10.0 mm Hg for both the SCCVII and the RIF-1 tumors. The data suggest that the majority of tumor cells with pO2 values below 5.0 mm Hg were clonogenic in the SCCVII tumors while only a small portion of tumor cells with pO2 below 5.0 mm Hg were clonogenic in the control RIF-1 tumors. Increased hypoxia caused by preirradiation of the tumor bed is a useful model in evaluating the efficacy of various treatments affected by tumor oxygenation levels. The model is also useful in evaluating techniques for measuring oxygen levels.

Hydralazine does not increase hypoxia in tumors growing in preirradiated tissue

There is a growing interest in the exploitation of hypoxia in solid tumors for therapeutic gain by the use of hypoxic cytotoxins and other agents. Tumor hypoxia can be greatly increased in a number of animal tumor models with the vasoactive drug hydralazine (HDZ), and in some cases this potentiates the effect of drugs that are selectively toxic to hypoxic cells. Our interest was to determine if HDZ would also increase tumor hypoxia in tumors growing in previously irradiated normal tissue- a situation such as might be found in the clinic with regrowing solid tumors after radiotherapy. SCCVII tumors in untreated mice were compared with tumors growing in a previously irradiated tissue with respect to their level of hypoxia in response to HDZ. HDZ increased tumor hypoxia in the tumors from unirradiated mice as measured by 14C-misonidazole binding. However, HDZ had little or no effect on tumor hypoxia in tumors growing in previously irradiated sites. We also showed that the pre-HDZ extent of hypoxia was higher in tumors growing in a previously irradiated tissue. This may in part explain the lack of effect of HDZ in these tumors. The lack of response of the tumors growing in irradiated sites suggests a limitation on the use of HDZ in combination with specific hypoxic cytotoxins or other chemotherapeutic drugs in the treatment of recurrent solid tumors. The data also show that if such tumors have an elevated hypoxic fraction relative to their counterparts growing in untreated sites, these tumors might be intrinsically more resistant to conventional radiotherapy, but, on the other hand, might be sensitive to bioreductive drugs and more likely to be radiosensitized by a hypoxic cell sensitizer.

Tumors growing in irradiated tissue: Oxygenation, metabolic state, and pH

Experimental tumors growing in irradiated tissue have been used to study the biological differences characteristic of locally recurrent tumors. Animal tumors were early generation isotransplants of a spontaneous fibrosarcoma in a C3Hf/Sed mouse, designated FSa-II. Since the hypoxic cell fraction of tumors growing in irradiated tissue is increased, these tumors are assumed to be metabolically deprived with hypoperfusion and acidosis. In this study we directly measured the oxygen partial pressure (pO 2) distribution, metabolic state, and pH of tumors growing in an irradiated tumor bed using oxygen sensitive electrodes and 31P-NMR. The results confirmed a three-fold increase in the number of pO 2 readings ≤ 2.5 mmHg and also showed increased acidosis with a 0.17 unit decrease in pH NMR. When tumors growing in pre-irradiated tissue reached ≈100 mm 3 in volume, a high frequency of gross and microscopic necrosis and hemorrhage was already observed. Consistent with these observations, the phosphocreatine/inorganic phosphate (PCr/P i) and nucleoside triphosphate/inorganic phosphate (NTP/P i) ratios were significantly lower in the tumors in a pre-irradiated bed compared to tumors in a non-irradiated bed (PCr/P i: 0.51 vs 0.79, p < 0.05; and NTP/P i,: 0.64 vs 0.93, p < 0.05). The longitudinal relaxation time (T 1) of P i was numerically shorter in control tumors (consistent with the better tissue oxygenation), but this did not reach statistical significance (2.09 ± .11 sec vs 2.25 ± .16 sec).

Thermotolerance in Preirradiated Intestine and Its Influence on Time-Temperature Relationships

The crypt compartment of mouse jejunum showed a transient increase in thermal susceptibility approximately 10 days after moderate X-ray doses to the abdomen (9-10 Gy). The increase in response was manifest as an increase in slope of the crypt dose-response curve but was limited to temperatures below 43 degrees C. As a result, the 43 degrees C inflexion in the Arrhenius plot (the relationship between treatment time and temperature) for thermal sensitivity of crypts was eliminated in preirradiated tissue, and the curve became monophasic over the range 42.0-44.5 degrees C. At temperatures below 42 degrees C, the curve again deviated. At supranormal temperatures of 42 degrees C and below, the durations of hyperthermia needed for measurable effect were sufficient to allow thermotolerance to be expressed within the heating period. Neither the threshold heating times nor this thermotolerance were affected by prior irradiation. In the temperature range 42-43 degrees C, an earlier development of thermotolerance could be demonstrated in control tissue by challenging with an acute high-temperature heat treatment. This thermotolerance was eliminated in preirradiated tissue, resulting in the apparent increase in sensitivity. The findings support the view that the complex nature of the time-temperature relationship seen in normal tissue in vivo is a manifestation of the ability of the tissue to progressively acquire a thermotolerant state during treatment at temperatures below approximately 43 degrees C, so that the "intrinsic" sensitivity is modulated while being assessed.

Erythrokinetics in mice bearing tumours in either preirradiated or unirradiated tissue.

Experiments were designed to clarify the causes of anaemia in hosts bearing tumours in either unirradiated or preirradiated tissue. Isotopic methods are described which enable the estimation of erythrocyte destruction and production rates, and the potential red cell life spans in tumour-bearing animals. In this experimental system, anaemia (a) is in large part due to accelerated random erythrocyte loss, (b) is exacerbated as tumours grow by a progressive reduction in the potential erythrocyte life span due to intrinsic erythrocyte defects, (c) is accompanied by an increase in erythrocyte production of six- to ten-fold and (d) is postponed in onset and decreased in magnitude by preirradiation of the tumour transplant site.