Treatment of perioperative swelling by rest, ice, compression, and elevation (RICE) without and with additional application of negative pressure (RICE+) in patients with a unilateral ankle fracture: study protocol for a monocentric, evaluator-blinded randomized controlled pilot trial.

BACKGROUND: Edema is commonly seen after surgical fixation of ankle fractures. Rest, ice, compression, and elevation (RICE) is an established combination to prevent swelling but hardly able to stimulate lymphatic resorption. Recently, an epicutaneously applied negative pressure suction apparatus (LymphaTouch®) has been introduced to stimulate lymphatic flow. While postoperative recovery, soft tissue, and osseous healing as well as functional outcome are probably linked to the amount of postoperative swelling, estimates on this relative to prevention (RICE) or prevention + stimulated resorption (RICE+) of fluid are scarce. METHODS AND ANALYSIS: This is a single-center, evaluator-blinded randomized pilot trial to investigate postoperative swelling in adults requiring surgical fixation of a closed unilateral ankle fracture. A total of 50 patients will be recruited and randomly assigned to RICE or RICE+ prior to surgery. All patients will undergo evaluator-blinded measurements of the ankle volume the day before surgery and subsequently from the evening of the 2nd postoperative day every 24 h until discharge. RICE will be initiated right after surgery and continued until discharge from the hospital in all patients. Additional application of negative pressure therapy (RICE+) will be initiated on the morning of the 2nd postoperative day and repeated every 24 h until the time of discharge from the hospital. Outcome measures are (i) the relative amount and the time course of the postoperative swelling, (ii) the demand for analgesic therapy (type and amount) together with the perception of pain, (iii) the rate of complications, and (iv) mobility of the ankle joint and the recovery of walking abilities during a 12-weeks follow-up period. Serum and urine samples taken prior to sugery and during postoperative recovery will allow to evaluate the ratio of naturally occurring stable calcium isotopes (δ44/42Ca) as a marker of skeletal calcium accrual. ETHICS AND DISSEMINATION: The protocol was approved by the institutional Ethics Committee (Rostock University Medical Center, Rostock, Germany) in accordance with the Declaration of Helsinki (approval number: A 2020-0092). The results of this study will be actively disseminated through scientific publications and conference presentations. TRIAL REGISTRATION: DRKS, DRKS00023739 . Registered on 14 December 2020.

[Fundamental concepts in the surgical treatment of fractures : Which implants should radiologists be familiar with?] / Grundkonzepte der operativen Frakturbehandlung : Welche Implantate sollte der Radiologie kennen?

BACKGROUND: Radiology is of enormous importance for orthopedic and trauma surgeons. There are almost no fields or subspecialties where adequate imagining is not a fundamental part of the diagnostic workup or therapy, especially in the treatment of fractures. OBJECTIVES: The aim of this article is to describe fundamental concepts in the treatment of fractures with regard to the various skeletal regions and implants, including their possibilities and limitations. METHODS: The article is mainly based on principles given by the AO (Arbeitsgemeinschaft für Osteosynthesefragen) Foundation in combination with our own experience related to shaft, close to joint, and joint fractures. RESULTS AND CONCLUSION: Basic concepts used in the surgical treatment of shaft, close to joints, and joint fractures as well as commonly used implants with regard to imaging required in this context without claiming to be exhaustive are reviewed. To improve outcome quality, close collaboration and direct communication between radiologists and orthopedic and trauma surgeons are required.

[Perioperative management of trauma surgery patients treated with direct oral anticoagulants]. / Perioperatives Management unfallchirurgischer Patienten unter Therapie mit direkten oralen Antikoagulanzien.

As the population gets older the prevalence of atrial fibrillation and venous thromboembolism also increases. Therefore, more patients require anticoagulation and currently direct oral anticoagulants (DOAC), such as dabigatran etexilate, apixaban, rivaroxaban and edoxaban are preferred to vitamin K antagonists (VKA), mainly because of the more favorable risk-benefit profile with respect to bleeding. Older patients in particular frequently present at the accident and emergency department due to falls and an increased risk of fractures. The perioperative management of these patients who are treated with DOACs is a challenge in the clinical routine and needs special consideration. This article discusses these issues in an interdisciplinary approach and develops strategies for the perioperative management of patients treated with DOACs and undergoing trauma or orthopedic surgery.

Overexpression of MicroRNA-1 in Prostate Cancer Cells Modulates the Blood Vessel System of an In Vivo Hen's Egg Test-Chorioallantoic Membrane Model.

BACKGROUND/AIM: In prostate cancer (PC), the formation of new blood vessels is stimulated by hypoxic conditions, androgens, and a number of molecular factors including microRNAs. MicroRNA-1 (miR-1) has been characterized in some tumor entities as anti-angiogenic, but this has not yet been investigated in PC. MATERIALS AND METHODS: PC cells stably overexpressing miR-1 (LNCaP-miR-1) were incubated on an in vivo hen's egg test-chorioallantoic membrane (HET-CAM) model and compared to maternal LNCaP cells. Cell growth, blood vessel organisation, and total blood vessel area were analysed. RESULTS: Both matrigel-embedded LNCaP and LNCaP-miR-1 cells formed compact tumor-like cell aggregates on the CAM of the HET-CAM model. Although not quantifiable, bleeding of the CAM and remodelling of the blood vessel network in the CAM indicated an influence of miR-1 on the vascular system. The statistically significant decrease in the total surface area of blood vessels in the visible CAM section to 79.4% of control cells demonstrated the antiangiogenic properties of miR-1 for the first time. CONCLUSION: MiR-1 had a tumor-suppressive and anti-angiogenic effect in an in vivo PC model. In the clinic, miR-1-mediated anti-angiogenesis would result in reduced tumor supply and increased hypoxic stress inside the tumor. Thus, miR-1 restoration by nucleic acid-based miR-1 mimetics would represent a promising option for future PC therapy.

Peroxiredoxin Expression of Human Osteosarcoma Cells Is Influenced by Cold Atmospheric Plasma Treatment.

BACKGROUND/AIM: To evaluate the potential involvement of redox-specific signalling pathways in cold atmospheric plasma (CAP)-induced apoptosis on human osteosarcoma cells. MATERIALS AND METHODS: Osteosarcoma cell lines were treated with CAP with or without antioxidative agents and seeded in cell culture plates. Cell proliferation was determined by counting viable cells. Carrier gas-treated cells served as control. Peroxiredoxin (PRX) 1-3 expression and secretion were assessed. RESULTS: CAP treatment exhibited strongly attenuated proliferation rates. This effect was significantly attenuated by the addition of N-acetylcysteine (NAC). CAP-treated cells exhibited an increase of PRX 1 and 2 10 sec after treatment. The ratio of oxidized to reduced PRX1 and PRX2 was significantly altered with increasing cellular concentration of the oxidized dimer. CONCLUSION: Antioxidant supplementation with NAC increases proliferation of CAP-treated osteosarcoma cells, implicating an involvement of redox signalling. Activation of PRX1 and -2 indicate CAP affects redox homeostasis.

New Treatment Options for Osteosarcoma - Inactivation of Osteosarcoma Cells by Cold Atmospheric Plasma.

BACKGROUND/AIM: Cold atmospheric plasma has been shown to inhibit tumor cell growth and induce tumor cell death. The aim of the study was to investigate the effects of cold atmospheric plasma treatment on proliferation of human osteosarcoma cells and to characterize the underlying cellular mechanisms. MATERIALS AND METHODS: Human osteosarcoma cells (U2-OS and MNNG/HOS) were treated with cold atmospheric plasma and seeded in culture plates. Cell proliferation, p53 and phospho-p53 protein expression and nuclear morphology were assessed. RESULTS: The treated human osteosarcoma cell lines exhibited attenuated proliferation rates by up to 66%. The cells revealed an induction of p53, as well as phospho-p53 expression, by 2.3-fold and 4.5-fold, respectively, compared to controls. 4',6-diamidino-2-phenylindole staining demonstrated apoptotic nuclear condensation following cold atmospheric plasma treatment. CONCLUSION: Cold atmospheric plasma treatment significantly attenuated cell proliferation in a preclinical in vitro osteosarcoma model. The resulting increase in p53 expression and phospho-activation in combination with characteristic nuclear changes indicate this was through induction of apoptosis.

Investigating the Mutagenicity of a Cold Argon-Plasma Jet in an HET-MN Model.

OBJECTIVE: So-called cold physical plasmas for biomedical applications generate reactive oxygen and nitrogen species and the latter can trigger DNA damage at high concentrations. Therefore, the mutagenic risks of a certified atmospheric pressure argon plasma jet (kINPen MED) and its predecessor model (kINPen 09) were assessed. METHODS: Inner egg membranes of fertilized chicken eggs received a single treatment with either the kINPen 09 (1.5, 2.0, or 2.5 min) or the kINPen MED (3, 4, 5, or 10 min). After three days of incubation, blood smears (panoptic May-Grünwald-Giemsa stain) were performed, and 1000 erythrocytes per egg were evaluated for the presence of polychromatic and normochromic nuclear staining as well as nuclear aberrations and binucleated cells (hen's egg test for micronuclei induction, HET-MN). At the same time, the embryo mortality was documented. For each experiment, positive controls (cyclophosphamide and methotrexate) and negative controls (NaCl-solution, argon gas) were included. Additionally, the antioxidant potential of the blood plasma was assessed by ascorbic acid oxidation assay after treatment. RESULTS: For both plasma sources, there was no evidence of genotoxicity, although at the longest plasma exposure time of 10 min the mortality of the embryos exceeded 40%. The antioxidant potential in the egg's blood plasma was not significantly reduced immediately (p = 0.32) or 1 h (p = 0.19) post exposure to cold plasma. CONCLUSION: The longest plasma treatment time with the kINPen MED was 5-10 fold above the recommended limit for treatment of chronic wounds in clinics. We did not find mutagenic effects for any plasma treatment time using the either kINPen 09 or kINPen MED. The data provided with the current study seem to confirm the lack of a genotoxic potential suggesting that a veterinary or clinical application of these argon plasma jets does not pose mutagenic risks.

Dorsal Skinfold Chamber Preparation in Mice: Studying Angiogenesis by Intravital Microscopy.

Intravital microscopy represents an internationally accepted and sophisticated experimental method to study angiogenesis, microcirculation, and many other parameters in a wide variety of neoplastic and nonneoplastic tissues. Since 1924, when the first transparent chamber model in animals was introduced, many other chamber models have been described in the literature for studying angiogenesis and microcirculation. Because angiogenesis is an active and dynamic process, one of the major strengths of chamber models is the possibility of monitoring angiogenesis in vivo continuously for up to several weeks with high spatial and temporal resolution. In addition, after the termination of experiments, tissue samples can be excised easily and further examined by various ex vivo methods such as histology, immunohistochemistry, and molecular biology. This chapter describes the protocol for the surgical preparation of a dorsal skinfold chamber in mice as well as the method to implant tumors in this chamber for further investigations of angiogenesis and other microcirculatory parameters. However, the application of the dorsal skinfold chamber model is not limited to the investigation of neoplastic tissues. To this end, the investigation of angiogenesis and other microcirculatory parameters of nonneoplastic tissues such as tendons, osteochondral grafts, or pancreatic islets has been an object of interest.

Cold Atmospheric Plasma Treatment Induces Anti-Proliferative Effects in Prostate Cancer Cells by Redox and Apoptotic Signaling Pathways.

One of the promising possibilities of the clinical application of cold plasma, so-called cold atmospheric plasma (CAP), is its application on malignant cells and cancer tissue using its anti-neoplastic effects, primarily through the delivery of reactive oxygen and nitrogen species (ROS, RNS). In this study, we investigated the impact of CAP on cellular proliferation and consecutive molecular response mechanisms in established prostate cancer (PC) cell lines. PC cells showed a significantly reduced cell growth following CAP treatment as a result of both an immediate increase of intracellular peroxide levels and through the induction of apoptosis indicated by annexin V assay, TUNEL assay, and the evaluation of changes in nuclear morphology. Notably, co-administration of N-acetylcysteine (NAC) completely neutralized CAP effects by NAC uptake and rapid conversion to glutathione (GSH). Vitamin C could not counteract the CAP induced effects on cell growth. In summary, relatively short treatments with CAP of 10 seconds were sufficient to induce a significant inhibition of cancer proliferation, as observed for the first time in urogenital cancer. Therefore, it is important to understand the mode of CAP related cell death and clarify and optimize CAP as cancer therapy. Increased levels of peroxides can alter redox-regulated signaling pathways and can lead to growth arrest and apoptosis. We assume that the general intracellular redox homeostasis, especially the levels of cellular GSH and peroxidases such as peroxiredoxins affect the outcome of the CAP treatment.

Cold atmospheric plasma in combination with mechanical treatment improves osteoblast growth on biofilm covered titanium discs.

Treatment of implants with peri-implantitis is often unsuccessful, because an instrumented implant surface and residual microbial biofilm impedes re-osseointegration. The application of cold atmospheric plasma (CAP) could be a simple and effective strategy to overcome the inherent problems of peri-implantitis treatment. CAP is able to destroy and eliminate bacterial biofilms. Additionally, it increases the wettability of titanium, which supports cellular attachment. In this study, the behaviour of osteoblasts on titanium discs was analysed after treatment of bacterial biofilms with CAP, brushing, or a combination of both. A human plaque biofilm was cultured on titanium discs. Treatment with a brush (BR), 1% oxygen/argon CAP (PL), or brushing combined with CAP (BR+PL) was used to eliminate the biofilm. Discs without biofilm (C), autoclaved biofilm (AUTO) and untreated biofilm (BIO) served as controls. Subsequently, human osteoblastic cell growth (MG-63) was observed after 1 and 24 h. Biofilm remnants on BR and PL impaired osteoblastic cell development, whereas the BR+PL provided an increased area of osteoblastic cells. A five-day cell growth was only detectable on BR+PL treated discs. The combination of established brushing and CAP application may be a promising strategy to treat peri-implantitis.

Celecoxib enhances radiation response of secondary bone tumors of a human non-small cell lung cancer via antiangiogenesis in vivo.

PURPOSE: Cyclooxygenase-2 (COX-2) inhibitors mediate a systemic antitumor activity via antiangiogenesis and seem to enhance the response of primary tumors to radiation. Radiosensitizing effects of COX-2 inhibition have not been reported for bone metastases. Therefore, the aim of this study was the investigation of the radiosensitizing effects of the selective COX-2 inhibitor celecoxib in secondary bone tumors of a non-small cell lung carcinoma in vivo. MATERIALS AND METHODS: Human A549 lung carcinomas were implanted into a cranial window preparation in male SCID mice (n = 24). Animals were treated with either celecoxib or radiation (7 Gy single photon dose) alone or a combination of celecoxib and radiation, respectively. Untreated animals served as controls. The impact of radiation and COX-2 inhibition on angiogenesis, microcirculation, and tumor growth was analyzed over 28 days by means of intravital microscopy and histological methods. RESULTS: Monotherapies with radiation as well as celecoxib had significant antitumor effects compared to untreated controls. Both therapies reduced tumor growth and vascularization to a similar extent. The simultaneous administration of celecoxib and radiation further enhanced the antitumor and antiangiogenic effects of single-beam radiation. With the combined treatment approach, tumor vascularization and tumor size were decreased by 57% and 51%, respectively, as compared to monotherapy with radiation. CONCLUSION: The combined application of radiation therapy and COX-2 inhibition showed synergistic effects concerning the inhibition of tumor growth and tumor angiogenesis. Therefore, the combination of radiation with COX-2 inhibitor therapy represents a promising approach to improve the therapeutic efficacy of radiotherapy of bone metastases.

The dorsal skinfold chamber: studying angiogenesis by intravital microscopy.

Intravital microscopy represents an internationally accepted and sophisticated experimental method to study angiogenesis, microcirculation, and many other parameters in a wide variety of neoplastic and nonneoplastic tissues. Since 1924, when the first transparent chamber model in animals was introduced, many other chamber models have been described in the literature for studying angiogenesis and microcirculation. Because angiogenesis is an active and dynamic process, one of the major strengths of chamber models is the possibility of monitoring angiogenesis in vivo continuously for up to several weeks with high spatial and temporal resolution. In addition, after the termination of experiments, tissue samples can be excised easily and further examined by various in vitro methods, such as histology, immunohistochemistry, and molecular biology. This chapter describes the protocol for the surgical preparation of a dorsal skinfold chamber in mice as well as the method to implant tumors in this chamber for further investigations of angiogenesis and other microcirculatory parameters. However, the application of the dorsal skinfold chamber model is not limited to the investigation of neoplastic tissues. To this end, the investigation of angiogenesis and other microcirculatory parameters of nonneoplastic tissues such as tendons, osteochondral grafts, or pancreatic islets have been objects of interest.

The cranial bone window model: studying angiogenesis of primary and secondary bone tumors by intravital microscopy.

The successful treatment of primary and secondary bone tumors in a huge number of cases remains one of the major unsolved challenges in modern medicine. Malignant primary bone tumor growth predominantly occurs in younger people, whereas older people predominantly suffer from secondary bone tumors since up to 85% of the most frequently occurring malignant solid tumors, such as lung, mammary, and prostate carcinomas, metastasize into the bone. It is well known that a tumor's course may be altered by its surrounding tissue. For this reason, reported here is the protocol for the surgical preparation of a cranial bone window in mice as well as the method to implant tumors in this bone window for further investigations of angiogenesis and other microcirculatory parameters in orthotopically growing primary or secondary bone tumors using intravital microscopy. Intravital microscopy represents an internationally accepted and sophisticated experimental method to study angiogenesis, microcirculation, and many other parameters in a wide variety of neoplastic and nonneoplastic tissues. Since most physiologic and pathophysiologic processes are active and dynamic events, one of the major strengths of chronic animal models using intravital microscopy is the possibility of monitoring the regions of interest in vivo continuously up to several weeks with high spatial and temporal resolution. In addition, after the termination of experiments, tissue samples can be excised easily and further examined by various in vitro methods such as histology, immunohistochemistry, and molecular biology.

Tyrosine kinase inhibitor SU6668 represses chondrosarcoma growth via antiangiogenesis in vivo.

BACKGROUND: As chondrosarcomas are resistant to chemotherapy and ionizing radiation, therapeutic options are limited. Radical surgery often cannot be performed. Therefore, additional therapies such as antiangiogenesis represent a promising strategy for overcoming limitations in chondrosarcoma therapy. There is strong experimental evidence that SU6668, an inhibitor of the angiogenic tyrosine kinases Flk-1/KDR, PDGFRbeta and FGFR1 can induce growth inhibition of various primary tumors. However, the effectiveness of SU6668 on malignant primary bone tumors such as chondrosarcomas has been rarely investigated. Therefore, the aim of this study was to investigate the effects of SU6668 on chondrosarcoma growth, angiogenesis and microcirculation in vivo. METHODS: In 10 male severe combined immunodeficient (SCID) mice, pieces of SW1353 chondrosarcomas were implanted into a cranial window preparation where the calvaria serves as the site for the orthotopic implantation of bone tumors. From day 7 after tumor implantation, five animals were treated with SU6668 (250 mg/kg body weight, s.c.) at intervals of 48 hours (SU6668), and five animals with the equivalent amount of the CMC-based vehicle (Control). Angiogenesis, microcirculation, and growth of SW 1353 tumors were analyzed by means of intravital microscopy. RESULTS: SU6668 induced a growth arrest of chondrosarcomas within 7 days after the initiation of the treatment. Compared to Controls, SU6668 decreased functional vessel density and tumor size, respectively, by 37% and 53% on day 28 after tumor implantation. The time course of the experiments demonstrated that the impact on angiogenesis preceded the anti-tumor effect. Histological and immunohistochemical results confirmed the intravital microscopy findings. CONCLUSION: SU6668 is a potent inhibitor of chondrosarcoma tumor growth in vivo. This effect appears to be induced by the antiangiogenic effects of SU6668, which are mediated by the inhibition of the key angiogenic receptor tyrosine kinases Flk-1/KDR, PDGFRbeta and FGFR1. The experimental data obtained provide rationale to further develop the strategy of the use of the angiogenesis inhibitor SU6668 in the treatment of chondrosarcomas in addition to established therapies such as surgery.

The selective Cox-2 inhibitor Celecoxib suppresses angiogenesis and growth of secondary bone tumors: an intravital microscopy study in mice.

BACKGROUND: The inhibition of angiogenesis is a promising strategy for the treatment of malignant primary and secondary tumors in addition to established therapies such as surgery, chemotherapy, and radiation. There is strong experimental evidence in primary tumors that Cyclooxygenase-2 (Cox-2) inhibition is a potent mechanism to reduce angiogenesis. For bone metastases which occur in up to 85% of the most frequent malignant primary tumors, the effects of Cox-2 inhibition on angiogenesis and tumor growth remain still unclear. Therefore, the aim of this study was to investigate the effects of Celecoxib, a selective Cox-2 inhibitor, on angiogenesis, microcirculation and growth of secondary bone tumors. METHODS: In 10 male severe combined immunodeficient (SCID) mice, pieces of A549 lung carcinomas were implanted into a newly developed cranial window preparation where the calvaria serves as the site for orthotopic implantation of the tumors. From day 8 after tumor implantation, five animals (Celecoxib) were treated daily with Celecoxib (30 mg/kg body weight, s.c.), and five animals (Control) with the equivalent amount of the CMC-based vehicle. Angiogenesis, microcirculation, and growth of A549 tumors were analyzed by means of intravital microscopy. Apoptosis was quantified using the TUNEL assay. RESULTS: Treatment with Celecoxib reduced both microvessel density and tumor growth. TUNEL reaction showed an increase in apoptotic cell death of tumor cells after treatment with Celecoxib as compared to Controls. CONCLUSION: Celecoxib is a potent inhibitor of tumor growth of secondary bone tumors in vivo which can be explained by its anti-angiogenic and pro-apoptotic effects. The results indicate that a combination of established therapy regimes with Cox-2 inhibition represents a possible application for the treatment of bone metastases.

Combined therapy with direct and indirect angiogenesis inhibition results in enhanced antiangiogenic and antitumor effects.

The multifaceted nature of the angiogenic process in malignant neoplasms suggests that protocols that combine antiangiogenic agents may be more effective than single-agent therapies. However it is unclear which combination of agents would be most efficacious and will have the highest degree of synergistic activity while maintaining low overall toxicity. Here we investigate the concept of combining a "direct" angiogenesis inhibitor (endostatin) with an "indirect" antiangiogenic compound [SU5416, a vascular endothelial growth factor receptor 2 (VEGFR2) receptor tyrosine kinase (RTK) inhibitor]. These angiogenic agents were more effective in combination than when used alone in vitro (endothelial cell proliferation, survival, migration/invasion, and tube formation tests) and in vivo. The combination of SU5416 and low-dose endostatin further reduced tumor growth versus monotherapy in human prostate (PC3), lung (A459), and glioma (U87) xenograft models, and reduced functional microvessel density, tumor microcirculation, and blood perfusion as detected by intravital microscopy and contrast-enhanced Doppler ultrasound. One plausible explanation for the efficacious combination could be that, whereas SU5416 specifically inhibits vascular endothelial growth factor signaling, low-dose endostatin is able to inhibit a broader spectrum of diverse angiogenic pathways directly in the endothelium. The direct antiangiogenic agent might be able to suppress alternative angiogenic pathways up-regulated by the tumor in response to the indirect, specific pathway inhibition. For future clinical evaluation of the concept, a variety of agents with similar mechanistic properties could be tested.

Reperfusion injury in free bone grafts after tourniquet-induced ischemia.

For successful revascularization, incorporation, and survival of fracture fragments or free nonpedicled bone grafts, the first days after surgery seem to be most critical. Using intravital fluorescence microscopy, the effects of 3 hours of tourniquet-induced ischemia were assessed on newly formed angiogenic microvessels 4, 8, and 12 days after isogeneic transplantation of metatarsal bones into dorsal skin fold chambers of adult inbred C57BL/6 mice. The microvascular perfusion index of newly formed microvessels was quantified 30 and 120 minutes after initiation of reperfusion. Four days after transplantation, a complete breakdown of the perfusion of all grafts was found accompanied by hemorrhage as a sign of microvascular damage. In contrast, at 8 and 12 days after transplantation a complete breakdown of graft perfusion rarely occurred. Therefore, the tourniquet-induced ischemia was most pronounced in early stages of development of angiogenic microvessels. These experimental data provide rationale for critical application of tourniquets in revision surgery shortly after initiation of fracture treatment or transplantation of free nonpedicled grafts so as not to compromise angiogenesis-induced tissue revascularization.

Acid production in glycolysis-impaired tumors provides new insights into tumor metabolism.

PURPOSE: Low extracellular pH is a hallmark of solid tumors. It has long been thought that this acidity is mainly attributable to the production of lactic acid. In this study, we tested the hypothesis that lactate is not the only source of acidification in solid tumors and explored the potential mechanisms underlying these often-observed high rates of acid production. EXPERIMENTAL DESIGN: We compared the metabolic profiles of glycolysis-impaired (phosphoglucose isomerase-deficient) and parental cells in both in vitro and two in vivo models (dorsal skinfold chamber and Gullino chamber). RESULTS: We demonstrated that CO(2), in addition to lactic acid, was a significant source of acidity in tumors. We also found evidence supporting the hypothesis that tumor cells rely on glutaminolysis for energy production and that the pentose phosphate pathway is highly active within tumor cells. Our results also suggest that the tricarboxylic acid cycle is saturable and that different metabolic pathways are activated to provide for energy production and biosynthesis. CONCLUSIONS: These results are consistent with the paradigm that tumor metabolism is determined mainly by substrate availability and not by the metabolic demand of tumor cells per se. In particular, it appears that the local glucose and oxygen availabilities each independently affect tumor acidity. These findings have significant implications for cancer treatment.