Hcc70

MDA-MB-231 and HCC70 cells from ATCC (Manassas, VA) were genetically engineered to express mWasabi-luciferase protein [24 (link)]. The Lenti-X™-293T cells were from Takara Bio (Takara Bio, San Jose, CA). Lenti-X™-293T and MDA-MB-231 were maintained in DMEM (Gibco; Thermo Fisher Scientific, MA), while HCC70 was cultured in RPMI1640 (Gibco). CAFs were isolated from BCA patients following the protocol approved by Siriraj Institutional Review Board (COA no. Si 329/2017). CAFs and normal fibroblasts (NFs) [normal breast fibroblast (BNF) and human dermal fibroblast (HDF)] were cultured in DMEM/F12 (Gibco). All complete media was supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin (Sigma-Aldrich, MA) and incubated with cells at 37˚C with 5% CO_2.

The following cell lines were obtained from the American Type Culture Collection (ATCC): i) TNBC lines MDA-MB-157, MDA-MB-436, MDA-MB-468, HCC70, BT-549, ii) ER+ /HER2/neu negative cell lines T-47D, ZR-75-1, MCF-7, MDA-MB-415, HCC1428, BT-483, iii) ER⁺/ Her2/neu⁺ cell lines BT474, and MDA-MB-361 and iv) ER-/Her2/neu⁺ cell lines AU565, HCC1954, and SKBR3. All cells except HS578T were cultured in DMEM-F12 containing 10% FBS. The TNBC line, HS578T also obtained from ATCC, was grown in DMEM with reduced NaHCO₃ (ATCC) containing 0.1 mM insulin and 10% FBS. The ER⁺/ Her2/neu-overexpressing MCF7 (MCF7-Her18) cells were a kind gift from Dr. Elizabeth Mittendorf. All the cell lines used in here were strictly with in ten passages after buying from ATCC and thus were not authenticated again.

BT-20, HCC70, MDA-MB-468, BT-549, MDA-MB-231, MDA-MB-436, and MCF-10A cells were purchased from the ATCC (Manassas, VA, USA). SUM159 cells were purchased from Astrerand (now BioIVT, Westbury, NY, USA) and iMEC cells were provided by Dr. Elizabeth Alli in the Department of Cancer Biology at Wake Forest School of Medicine. Cell lines were expanded, and low passage stocks were stored in liquid nitrogen and maintained by the Wake Forest Comprehensive Cancer Center Cell Engineering Shared Resource. Cell lines and growth media are listed in Table 1.
All cells were verified to be free from mycoplasma contamination by routine testing using the MycoAlert Mycoplasma Detection Kit (Lonza, Morristown, NJ, USA). Cells were passaged and medium was changed twice weekly. Cell monolayers were grown on tissue culture treated plastics purchased from Corning Life Sciences (Corning, NY, USA) or on glass coverslips (Warner Instruments Corporation, Hamden, CT, USA). For live fluorescence imaging studies, cells were grown in 8-well chamber slides (EMD Millipore, Burlington, MA, USA). Cells were maintained in culture for no longer than 4 months before new cultures were established from low-passage frozen stocks.

The HCT116, HT29, MDA-MB-231, SW480, CaCO2, HCC70, MCF7, AGS, and NCI-N87 cell lines were purchased from ATCC. The MDA-MB-231-SMYD3-KO and HCT116-SMYD3-KO cell lines were generated using the CRISPR/Cas9 technology. The U2OS cell line was kindly provided by Prof. Jeremy Stark.
The HCT116-OXA-R cell line was established from the HCT116 parental cell line by continuous exposure to oxaliplatin with stepwise increasing concentrations ranging from 1 μM to 10 μM over approximately 6 months. The CRC cell lines HCT116, HT29, SW480, and CaCO2, and the BC cell lines MDA-MB-231 and MCF7, and the U2OS DR-GFP cell line were cultured in DMEM high glucose without pyruvate (41965–039, Gibco) with 10% FBS (A5256701, Gibco) and 100 IU/ml penicillin–streptomycin (15140–122, Gibco). The BC cell line HCC70 and the GC cell lines AGS and NCI-N87 were cultured in RPMI high glucose without pyruvate (21875–034, Gibco) with 10% FBS (A5256701, Gibco) and 100 IU/ml penicillin–streptomycin (15140–122, Gibco). All cell lines were tested to be mycoplasma-free (117048; Minerva Biolabs) multiple times throughout the study. All cell cultures were maintained in a humidified incubator at 37 °C and 5% CO2.

BC cell lines (EVSA-T (DSMZ), HCC70 (ATCC) and ZR-75-1 (ATCC)) were cultured in RPMI (ThermoFisher) supplemented with 10% FBS (ThermoFisher) and 1% GlutaMax (ThermoFisher). HEK293T cells were cultured in DMEM supplemented with, 10% FBS and 1% GlutaMax (ThermoFisher). All cell lines used in this study were authenticated using STR fingerprinting and negative for mycoplasma. Cells were cultured at 37 °C under 5% CO₂. All compounds used in this study (AZD8186, capivasertib, AZD5991, Rapamycin, AZD2014, AZD8835, AZD8931, AZD4320, AZD9496) were synthesized at AstraZeneca. For in vitro experiments, all inhibitors were dissolved in DMSO to a concentration of 10 mM and stored at −80 ^oC. Unless stated otherwise, EVSA-T cells were treated 250 nM AZD8186, 1 µM capivasertib and 50 nM AZD5991; HCC70, 100 nM AZD8186, 500 nM capivasertib and 200 nM AZD5991; ZR-75-1, 100 nM AZD8186 and 500 nM capivasertib.

The human BC cell lines, MCF7, BT-549, MDA-MB-453, MDA-MB-231, MDA-MB-468, and HCC70, were procured from ATCC (Manassas, VA). All the cell lines were maintained in their required Dulbecco's Modified Eagle Medium (DMEM) (GE Healthcare Life Sciences, Logan, Utah), or Minimum Essential Medium (MEM) (GE Healthcare Life Sciences) supplemented with 5 % or 10 % fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), penicillin (100 units/ml) and streptomycin (100 μg/ml) (Invitrogen, Carlsbad, CA) in a humidified atmosphere of 5 % CO₂ at 37 °C. All the cell lines were tested periodically for mycoplasma and determined to be free from infection. Serum specimens from breast cancer patients of AA and CA racial backgrounds were obtained through Institutional Biobank under the Institutional Review Board-approved protocol.

All experiments were performed using NSP and SP of MCF-7 cell line, MDA-MB-231 and HCC-70 BC cell lines. MCF-7, MDA-MB-231 and HCC-70 were purchased from ATCC (Manassas, USA). NSP and SP cells from MCF-7 cells were isolated and characterized in our core facilities. Details of culture conditions and procedures of these cell lines were described previously^{26 (link)}. Unless stated otherwise, all reagents and chemicals were purchased from Sigma-Aldrich (St. Louse, MO, USA). Human recombinant CCN5 protein (hrCCN5) was obtained from PeproTech (Rocky Hill, NJ, USA) and purity was tested after every purchased using Western blot analysis (see Figure S1). CCN5 antibody was generated in our laboratory^{50 (link)}. Fetal bovine serum (FBS) was obtained from ATCC (Manassas, VA, USA). Soft agar assay kit was purchased from Cell Biolabs, Inc. (San Diego, CA, USA). CD44 monoclonal antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), CD24 from Santa Cruz Biotechnology (Dallas, TX, USA), E-cadherin from BD Biosciences (Franklin Lakes, NJ, USA); Vimentin from Thermo Fisher Scientific (Waltham, MA, USA). The dilution of the antibodies was used as per manufacturer’s recommendation.