Lapatinib

Tumor tissue from the CX17 patient was obtained following surgery and implanted underneath the renal capsules of female BALB/c nude mice as described above. After engraftment and tumor mass formation, the tumors were passaged and expanded by 5 generations (M5) with 2 cohorts each consisting of 10 mices. After implantation (8 weeks), the established tumors (N = 10 for each tumor sample) were treated with the following regimens: trastuzumab (Roche Genentech, San Francisco, CA, USA) 10 mg/kg twice weekly and lapatinib (LC Laboratories, Woburn, MA, USA) 100 mg/kg daily. After 3 weeks of treatment, the tumors grown under the subrenal capsule were harvested and weighed.

ATRA was from Sigma (St Louis, MI, USA) and Lapatinib from LC Laboratories (Woburn, MA, USA). SKBR3, MD-MB157, MDA-MB231, MCF-7 and MDA-MB435 cells (ATCC, American-Type-Culture-Collection) were seeded at 3 × 10⁵ cells/cm², cultured and treated as described [1 (link)].

Ganetespib was purchased from Medkoo Biosciences, Inc. (Chapel Hill, NC). Lapatinib was purchased from LC Laboratories (Woburn, MA). Cycloheximide was purchased from Sigma-Aldrich (St. Louis, MO). Primary antibodies specific to cleaved PARP, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, Bcl-2, ErbB2, phospho-ErbB2 (pErbB2), Akt, pAkt, Erk2, pErk1/2, mTOR, pmTOR, Cyclin B1, Src, pSrc, Bad, pBad, GSK3, and pGSK3 were purchased from Cell Signaling Technology (Danvers, MA). Primary antibodies against CDK1, Cyclin D1, p27, caspase 8, caspase 9, HSP90, HSP70, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary mouse and rabbit antibodies were purchased from Thermo Scientific (Rockford, IL).

Acini cultures were performed as described^{52 (link), 53 (link)}. eCCs (5 × 10⁴) were seeded in 400 μl Assay Medium in 8-well chamber slides coated with 40 μl of Matrigel (Corning). Acini formed at an efficiency of around 30 acini/1 × 10⁴ mammary epithelial cells plated. For macrophage co-cultures, primary tissue macrophages were added at a ratio of 500 per 1 × 10⁴ eCCs seeded to 5-day-old acini cultures. For inhibitory treatments, 5-day-old acini cultures were treated for 24 h with 1 μM lapatinib (LC Laboratories), 2 μM IKK inhibitor^{38 (link)} (generous gift from Dr. Albert Baldwin), 1 μM CCR2 inhibitor RS504393 (Tocris Bioscience) or DMSO as vehicle control. Cultures were then fixed for IF with 4% PFA. Mammosphere cultures were prepared as described^{53 (link)}. To prepare CM, 5-day-old mammosphere culture were plated in serum-free DMEM medium and CM was harvested after 24 h. ELISA detection for CCL2 was done following the protocol provided by the manufacturer (catalog number: MJE00, R&D systems).