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Quantitative assessment of hypoxia subtypes in microcirculatory supply units of malignant tumors Using (immuno-)fluorescence techniques

Quantitative Erfassung verschiedener Hypoxieformen in mikrozirkulatorischen Versorgungseinheiten maligner Tumoren mit Hilfe von (Immun-)Fluoreszenztechniken

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Abstract

Background and Purpose:

Hypoxia is a characteristic of tumors, is known to increase aggressiveness, and causes treatment re-sistance. Traditional classification suggests two types of hypoxia: chronic and acute. Acute hypoxia is mostly caused by transient disruptions in perfusion, while chronic hypoxia is caused by diffusion limitations. This classification may be insufficient in terms of pathogenetic and pathophysiological mechanisms. Therefore, we quantified hypoxia subtypes in tumors based on (immuno-)fluorescent marker distribution patterns in microcirculatory supply units (MCSUs).

Material and Methods:

Cryosections from hSCC lines (SAS, FaDu, UT-SCC-5, UT-SCC-14, UT-SCC-15) were analyzed. Hypoxia was identified by pimonidazole, perfusion by Hoechst 33342, and endothelial cells by CD31. The following patterns were identified in vital tumor tissue: (1) normoxia: Hoechst 33342 fluorescence around microvessels, no pimonidazole, (2) chronic hypoxia: Hoechst 33342 fluorescence around microvessels, pimonidazole distant from microvessels, (3) acute hypoxia: no Hoechst 33342 fluorescence around microvessels, pimonidazole in immediate vicinity of microvessels, and (4) hypoxemic hypoxia: Hoechst 33342 fluorescence and pimonidazole directly around microvessels.

Results:

Quantitative assessment of MCSUs show predominance for normoxia in 4 out of 5 tumor lines (50.1–72.8%). Total hypoxia slightly prevails in UT-SCC-15 (56.9%). Chronic hypoxia is the dominant subtype (65.4–85.9% of total hypoxia). Acute hypoxia only accounts for 12.9–29.8% and hypoxemic hypoxia for 1.2–6.4% of total hypoxia. The fraction of perfused microvessels ranged from 82.5–96.6%.

Conclusion:

Chronic hypoxia is the prevailing subtype in MCSUs. Acute hypoxia and hypoxemic hypoxia account for only a small fraction. This approach enables assessment and recognition of different hypoxia subtypes including hypoxemic hypoxia and may facilitate methods to (clinically) identify and eliminate hypoxia.

Zusammenfassung

Hintergrund und Ziel:

Hypoxie ist ein Charakteristikum solider Tumoren, führt zur Tumorprogression und Therapieresistenz. Traditionell werden chronische und akute Hypoxie unterschieden. Erstere beruht vor allem auf Diffusionslimitierungen, letztere bevorzugt auf Perfusionsstörungen. Diese Klassifizierung reicht nicht aus, um pathogenetische und pathophysiologische Mechanismen weiter aufzuklären. Deshalb werden Hypoxiesubtypen in mikrozirkulatorischen Versorgungseinheiten (MCSUs) mit Hilfe von (Immun-)Fluoreszenztechniken identifiziert.

Material und Methoden:

Gefrierschnitte von xenotransplantierten menschlichen Plattenepithelkarzinomen (SAS, FaDu, UT-SCC-5, UT-SCC-14, UT-SCC-15) werden nach Pimonidazol-Färbung zur Hypoxiemarkierung, Hoechst-33342-Fluoreszenz zum Perfusionsnachweis und CD31-Gefäßdarstellung untersucht. Folgende Muster können in vitalem Gewebe nachgewiesen werden: (1) Normoxie: Hoechst-33342-Fluoreszenz um Gefäße, keine Pimonidazol-Anfärbung; (2) chronische Hypoxie: Hoechst-33342-Fluoreszenz in direkter Gefäßnähe, Pimonidazol in einer gewissen Distanz zu den Gefäßen; (3) akute Hypoxie: Hoechst-33342-Flu-oreszenz fehlt, Pimonidazol in unmittelbarer Gefäßnachbarschaft und (4) hypoxämische Hypoxie: Hoechst-33342-Fluoreszenz und Pimonidazol in direkter Gefäßnachbarschaft.

Ergebnisse:

Die Verteilungsmuster von Hoechst, Pimonidazol und CD31 in den MCSUs weisen darauf hin, dass in 4 der 5 Tumorlinien normoxische Areale überwiegen (50,1–72,8%). Lediglich UT-SCC-15-Tumoren sind überwiegend (56,9%) hypoxisch. Die Analyse der Hypoxiesubtypen zeigt, dass chronische Hypoxie eindeutig überwiegt (65,4–85,9% der Gesamthypoxie). Auf die akute Hypoxie entfallen lediglich 12,9–29,8% der Gesamthypoxie. Der Anteil der hypoxämischen Hypoxie ist am kleinsten (1,2–6,4% der Gesamthypoxie). Die Fraktion der perfundierten Gefäße beträgt 82,5–96,6%.

Schlussfolgerung:

Chronische Hypoxie herrscht in mikrozirkulatorischen Versorgungseinheiten der untersuchten Plattenepithelkarzinome vor. Akute und hypoxämische Hypoxie spielen nur eine untergeordnete Rolle. Der experimentelle Ansatz erlaubt erstmalig die Erfassung der hypoxämische Hypoxie im Tumorgewebe und ermöglicht eine Differenzierung verschiedener Hypoxiesubtypen. Die beschriebene Methode könnte die (quantitative) Detektion hypoxischer Areale und klinisch relevante Maßnahmen zur Verbesserung des Oxygenierungsstatus erleichtern.

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Authors and Affiliations

  1. Department of Radiotherapy and Radiation Oncology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany

    Constantin-Alin Maftei, Christine Bayer Ph.D., Kuangyu Shi, Sabrina T. Astner & Peter Vaupel

  2. Department of Radiotherapy and Radiation Oncology, Klinikum rechts der Isar, Technical University of Munich, Ismaninger Str. 22, 81675, München, Germany

    Christine Bayer Ph.D.

Authors
  1. Constantin-Alin Maftei
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  2. Christine Bayer Ph.D.
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  3. Kuangyu Shi
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  4. Sabrina T. Astner
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  5. Peter Vaupel
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Correspondence to Christine Bayer Ph.D..

Additional information

Presented in part at the 16th Jahreskongress der Deutschen Gesellschaft für Radioonkologie (DEGRO), Magdeburg, Germany, 2010

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Maftei, CA., Bayer, C., Shi, K. et al. Quantitative assessment of hypoxia subtypes in microcirculatory supply units of malignant tumors Using (immuno-)fluorescence techniques. Strahlenther Onkol 187, 260–266 (2011). https://doi.org/10.1007/s00066-010-2216-0

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  • DOI: https://doi.org/10.1007/s00066-010-2216-0

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