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268 Chapter 6: Breast Cancer
Tumor cell clonogenic assay
The mononuclear cell fraction was placed in medium ready for plating. In some
cases where multiple PBPC specimens were obtained, the specimens were pooled
and processed as a single specimen. A total of 5.0XI0 5
to 1.0X10 6
mononuclear
cells per mL were plated in triplicate in 35 mm 2
grid-bottomed petri dishes (Nunc,
Naperville, IL) in a soft agar-based medium consisting of final concentration of 15%
Iscove's modified Dulbecco's medium (IMDM) (Sigma), 10% fetal bovine serum
(Sigma), 0.3% agar solution (Sigma), 2 pg/mL human recombinant epidermal
growth factor (Collaborative Research, Bedford, MA), and 10 pg/mL human
recombinant insulin-like growth factor I (Collaborative Research). Negative control
plates consisted of medium without supplemental growth factors and PBPC
specimens from patients with nonepithelial tumors (e.g., normal donors, neurob­
lastoma, etc.). All plates were incubated in a humidified chamber at 37°C with 7.5%
C0 2 for 10-14 days. An inverted phase contrast microscope was used to count
tumor colonies (>25 cells).
Tumor colony identification
To definitively distinguish tumor colonies from normal hematopoietic
colonies, agar plates were floated onto 2- X 3-inch microscope slides and air-dried
at 4°C. Initially, in situ tumor colony verification was performed using immunofluorescence
staining with FITC-labeled anti-cytokeratin mAb SB-3 (Caltag, San
Francisco, CA) (Fig. 1). In certain cases, granulocyte-macrophage colony-forming
unit (CFU-GM) colony verification was performed using immunofluorescence
staining with rhodamine-labeled anti-CD 11 mAb (Dako, Carpenteria, CA). Later,
an immunocytochemical method was developed to verify and count the number of
tumor colonies. Slides were placed into cold storage at -70°C. The patient
specimens were then removed from storage and tested for immunoreactivity.
Immunostaining was accomplished using the alkaline phosphatase-based
technique. Slides were baked in an oven, fixed in a paraformaldehyde solution at
4°C for 30 minutes, and washed thoroughly in PBS with 1% triton (Gibco). After
fixation, slides were incubated with agarase followed by anti-breast carcinoma
antibodies for 45 minutes at room temperature. Biotek immunoreagents for
alkaline phosphatase staining were performed as per protocol. The specimen was
then incubated with Ehrlich's hematoxylin counterstain and coverslipped with
mounting medium.
The number of tumor colonies per plate was then recorded as identified by the
above methods. PBPC specimens were excluded from analysis if the cell viability
was