The Indiana University Animal Care and Use Committee approved the use of animals in this study and all procedures were performed as per NIH guidelines. Transformed cells with 50% basement membrane matrix (BME) type 3 (3632-005-02, Trevigen) (total 100 µL volume) were implanted into the mammary fat pad of 5–6-week-old female NSG (NOD/SCID/IL2Rgnull) mice. A 60-days slow release 17β-estradiol (0.72 mg) pellet (SE-121, Innovative Research of America) was implanted at the time of mammary fat pad injection. Tumor growth was measured weekly and tumor volume was calculated using the formula- sagittal dimension (mm) × [cross dimension (mm)]2/243 (link). The maximal tumor burden permitted by the Indiana University Animal Care and Use Committee is 2 cm × 2 cm. The maximal tumor burden was never exceeded during this study. After 2–4 months, tumors and lungs were collected and processed for hematoxylin and eosin (H&E), ERα, GATA3, FOXA1, EpCAM (MA5-29698, Invitrogen,1:500), CK5/6 (IR 780, Dako, Ready-to-use), CK8 (35bH11, N1560, Dako, 10 µg/ml), CK14 (LL002, ab7800, Abcam, 1 µg/ml), and CK19 (IR 615, Dako, Ready-to-use) staining43 (link).
Ir 615
Manufactured by Agilent Technologies
The IR 615 is a Fourier Transform Infrared (FTIR) spectrometer manufactured by Agilent Technologies. It is a compact and versatile instrument designed for a wide range of infrared spectroscopy applications. The IR 615 provides high-quality infrared spectra for the analysis of organic and inorganic materials.
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2 protocols using ir 615
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Xenograft Model of Breast Cancer
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Tumor Growth Dynamics and Marker Profiling
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Tumor cells (10,000, 50,000, 100,000, 1 × 106, or 5 × 106, total 100-μl volume) processed at 3% O2 and 21% O2 were implanted into the fifth inguinal mammary fat pad of each 6- to 8-week-old syngeneic FVB/N female mice without expanding in culture. Tumor growth was measured weekly, and tumor volume was calculated as described previously (55 (link)). Tumor tissues were harvested and divided into two fractions at their respective 3% O2 or ambient air. One fraction was collected for immunohistochemistry, and the other fraction was dissociated using a 10% collagenase/hyaluronidase enzyme mixture and processed at their respective 3% O2 or ambient air, and cells were either processed immediately for CSC marker profiles, reinjected into animals, or cultured at 5% O2 and 21% O2.
H&E, LGR5, TSPAN8, EpCAM, CK19, and CK14 immunostaining was performed at the CLIA (clinical laboratory improvement amendments)-certified Indiana University Health Pathology Laboratory, and the whole-slide digital imaging system of Aperio (ScanScope CS) was used for imaging according to a protocol described previously (55 (link)). The following antibodies were used: LGR5 (ab75732, Abcam), TSPAN8 (PA5-75311, Invitrogen), EpCAM (MA5-29698, Invitrogen), CK19 (IR 615, Dako), and CK14 (LL002, ab7800, Abcam).
H&E, LGR5, TSPAN8, EpCAM, CK19, and CK14 immunostaining was performed at the CLIA (clinical laboratory improvement amendments)-certified Indiana University Health Pathology Laboratory, and the whole-slide digital imaging system of Aperio (ScanScope CS) was used for imaging according to a protocol described previously (55 (link)). The following antibodies were used: LGR5 (ab75732, Abcam), TSPAN8 (PA5-75311, Invitrogen), EpCAM (MA5-29698, Invitrogen), CK19 (IR 615, Dako), and CK14 (LL002, ab7800, Abcam).
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