The triple negative patient-derived tumor, designated as TU-BCx-2 K1, was acquired in collaboration with the Louisiana Cancer Research Consortium Biospecimen Core and were processed in compliance with NIH regulations and institutional guidelines, and approved by the Institutional Review Board at Tulane University. The TU-BcX-2 K1 model was derived from a biopsy specimen from an African-American patient who had not yet initiated a chemotherapy regimen. For comparison of tumor growth, we also mention TU-BcX-2O0, a claudin-low TNBC PDX model established from an African American patient; TU-BcX-2O0 was also treatment naïve. TU-BCx-2 K1 was established and propagated in immunocompromised SCID/Beige mice. SCID/Beige
(CB17.Cg-
PrkdcscidLyst bg/Crl
) 4–6 weeks of age were purchased from Charles River and were used to prevent rejection of the xenografted human tumors. The autosomal recessive SCID (Prkdc
scid) mutation results in severe combined immunodeficiency affecting both the B and T lymphocytes. The autosomal recessive beige (Lyst
bg) mutation results in defective natural killer (NK) cells. Mice acclimated for 5–7 days in sterile laboratory cages with appropriate bedding material and autoclaved food and water before experiments were performed. Tumor tissues from patients were dissected into 3 × 3 mm
3 pieces under aseptic sterile conditions, coated with full factor Matrigel™ (Cat No. 354234, Fisher Scientific, Waltham, MA, USA) and implanted bilaterally into the mammary fat pads (MFPs) of SCID/Beige mice under isoflurane and oxygen. When tumors were large enough to be palpable, tumors were measured using a digital caliper. When tumor volume reached 750–1000 mm
3, tumors were passaged, or serially transplanted, into new mice. For serial transplantation, the mice with the large PDX tumors were euthanized by CO
2 and cervical dislocation, and tumors were removed, dissected to 3 × 3 mm
3 pieces and coated in full factor Matrigel™. The coated tumors were then implanted bilaterally into new mice that were anesthetized using a mix of isoflurane and oxygen delivered by mask. Before surgery, mice were given Meloxicam (5 mg/kg/day, for 3 days post-surgery) for pain and mice were monitored for 3 days for evidence of distress; if distress was observed, mice were euthanized. For ex vivo analysis, TU-BCx-2 K1 explants were collected, and RNA was extracted using enzymatic digestions using QIAzol Lysis Reagent (Cat No. 79306; Qiagen, Valencia, CA, USA) and mechanical disruption using scissors. Total RNA was isolated, and cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). cDNA was analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR). Primers (Invitrogen, Carlsbad, CA) were generated with sequences as follows:
β-actin F-5′- GGCACCCAGCACAATGAAGA-3′;
β-actin R-5′- ACTCCTGCTTGCTGATCCAC -3′;
CDH1 F-5′-AGGTGACAGAGCCTCTGGATAGA-3′,
CDH1 R-3′-TGGATGACACAGCGTGAGAGA-3′;
CDH2 F-5′-GCCCCTCAAGTGTTACCTCAA-3′,
CDH2 R-5′- AGCCGAGTGATGGTCCAATTT-3′;
VIM F-5′-CGTCCACCCGCACCTACAGC-3′,
VIM R-5′-GCCAGCGAGAAGTCCACCGAG-3′;
CD24 F-5′- TGCTCCTACCCACGCAGATT-3′,
CD24 R-5′- GGCCAACCCAGAGTTGGAA-3′;
cFOS F-5′- GAATGCGACCAACCTTGTGC-3′;
cFOS R-5′- AGGGATCAGACAGAGGGTGT-3′;
SNAI1 F-5′- AGCCGTGCCTTCGCTGACC-3′;
SNAI1 R-5′- GGACTCTTGGTGCTTGTGGAGC-3′;
FRA1 F-5′- CGAAGGCCTTGTGAACAGAT-3′,
FRA1-R-5′- CTGCAGCCCAGATTTCTCA-3′;
TWIST F-5′- TGTCCGCGTCCCACTAGC-3′
TWIST R-5′- TGTCCATTTTCTCCTTCTCTGGA-3′;
SLUG F-5′- TGTTGCAGTGAGGGCAAGAA-3′,
SLUG R-5′- GACCCTGGTTGCTTCAAGGA-3′;
cMYC F-5′- CAGCGGGCGGGCACTTTG-3′,
cMYC R-5′- AGAGAAGCGGGTCCTGGCA-3′. qRT-PCR was conducted as previously published [
24]. Data represented as normalized fold expression compared with DMSO control of biological triplicate samples ± S.E.M.