Detection and measurement of phosphorylated signaling proteins in solid tumor fine-needle biopsies by laser-scanning cytometry

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Background:The application of molecular targeted therapeutics is expected to yield a modulation of the respective cellular signaling pathway(s) which is usually monitored by sequential biopsies. However, fine-needle sampling (FNS) is a better tolerated, minimally invasive technique that can be repeated at numerous points during the clinical or experimental administration of a drug. Small volumes and paucicellularity of fine-needle samples previously often precluded a comprehensive analysis by flow cytometry as well as other techniques.We here describe the image based detection of phosphorylated signaling proteins in FNS specimens and the measurement of pathway activities by laser scanning cytometry (LSC), a slide-based technique that permits the acquisition of quantitative data as well as morphological information.Design:Fine-needle samples were obtained from s.c. xenografts of the cervical cancer cell line SiHa grown in SCID mice, using 21 Gauge needles and a non-aspiration technique (Zajdela). Specimens were directly expelled into formalin and used for tissue block preparations. Slides obtained from these specimens were used for the detection and quantification of signaling pathway activities based on phosphospecific labeling for S⁷²⁷-Stat3, Y⁷⁰⁵-Stat3 and S²⁷⁶-NFkappaB in immunofluorescence and immunohistochemistry. Analysis was performed using the LSC and iCys instrumentation (CompuCyte Corp., Cambridge, MA). Random (stereometric) and cell based sampling/contouring strategies were compared for chromatic labeling using the iCys instrumentation.Results:Phosphorylated Stat3 and NFkappaB showed a relatively homogeneous distribution throughout the viable tumor area which ensured representativeness of the obtained FNS specimens. Changes in phosphorylation state were observed due to delayed fixation and could be reproducibly measured based on fluorescent or chromatic labeling. Random and cell based analysis yielded comparable results. The accuracy of the measurement could be better controlled using the more advanced iCys instrumentation due to more precise selection of appropriate sample areas and better algorithms for spectral overlap compensation.Conclusions:FNS of solid tumors may be useful in anatomic sites where sampling for core-needle biopsies is not possible or not well tolerated. Instantaneous fixation of FNS specimens minimizes errors that may occur due to delayed tissue fixation. FNS can be used for biomarkers with a relatively homogeneous distribution throughout the tumor. Slide-based cytometry may be applied to estimate pathway activities before and after the application of molecular targeted agents.