Mycoalert

Murine Panc02-EpCAM, 4T1, T110299 and CT26-EpCAM have been previously described [41 (link)–43 ]. Murine LL/2 were purchased from the European Collection of Authenticated Cell Cultures (ECACC). The T110299 and LL/2 cell lines were modified to stably express full-length murine EpCAM (UNIPROT entry Q99JW5), to generate the cell lines T110299-EpCAM and LL/2-EpCAM. Human SUIT-2-MSLN have been previously described [41 (link)]. 293Vec-Galv, 293Vec-Eco, and 293Vec-RD114 have been previously described [44 (link)]. The virus producing cell lines 293Vec-Eco for anti-EpCAM-CAR-28z and 293Vec-RD114 for anti-MSLN-CAR-28z have been previously described [45 (link)]. 293Vec-RD114 for anti-MSLN-CAR-4-1BBz were generated as previously described [46 (link)]. All cell lines were cultured as previously described [41 (link), 42 (link)]. All cell lines were periodically tested for mycoplasma contamination with the commercial testing kit MycoAlert^TM (Lonza, Basel, Switzerland). Authentication of human cell lines by short tandem repeat DNA profiling was conducted in-house.

HEK293F cells were a gift from Nicola Burgess-Brown (University of Oxford). THP-1 cells were purchased from Sigma. A MycoAlert^TM (Lonza) kit was used to confirm freedom from Mycoplasma contamination. Cells were functionally authenticated by protein production and chemokine-induced migration as appropriate.

The cultured human epithelial breast cancer cell lines, MCF7 (estrogen receptor positive, HER2 negative) and BT474, were purchased from the American Type Culture Collection (ATCC, LGC Standards) and were authenticated by the vendor. Both cell lines were tested for mycoplasma contamination using MycoAlert^TM (Lonza) and Universal Mycoplasma Detection Kit (ATCC) and were found to be negative. MCF7 and BT474 cells were cultured at 37 °C with 5% CO₂ and maintained by regular passage in complete media consisting of Dulbecco’s Modified Eagle’s Media (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (GIBCO^®, Life Technology). When cells reached a confluency of 70–80%, they were collected and fluorescently labeled with CellTracker^TM Green CMFDA dye (Invitrogen, C7025) or CellMask^TM Orange plasma membrane stain (Invitrogen, C10045) for cell capture experiments. For in vivo cell capture, MCF7 cells purchased from ATCC were transfected with NucLight Red^TM (Essen BioScience, Ann Arbor, MI) according to manufacturer instructions (Supplementary Fig. 12).

Cell lines maintained in DMEM supplemented with 10% FBS were available from ATCC (HCC-1806, MDA-MB-231) and Creative Bioarray (CAL-51, SUM-159pt). Cell lines were authenticated by STR analyses and mycoplasma tested (MycoAlert^TM, Lonza) at regular intervals. Cells were maintained in a humidified incubator at 5% CO₂ and 37°C and hypoxic exposure was at either 1% or 0.5% (BakerRuskinn, InvivO₂). Normoxia was defined as 21% O₂ 5% CO₂. Acidosis media was prepared as previously [16] (link). Spheroid aggregation was initiated with 10,000 cells in ultra-low–adherent round-bottom 96-well plates followed by centrifugation at 2000 × g. Clonogenic assays were performed as previously [16] (link). To knockdown SLC4A4 or SLC4A5 in HCC-1806, CAL-51, and MDA-MB-231, shRNA lentivirus was purchased from Sigma-Aldrich (Supplementary Table 2). Cells were grown under Puromycin (Gibco) selection (HCC-1806 and MDA-MB-231 2 μg/mL; CAL-51, 10 μg/mL) to select shRNA expressing cells. Experiments were optimised and normalised by cell number at the end of experiments.

Human U-2OS, MG-63 and SaOS-2 osteosarcoma cell lines were obtained from ATCC (Molsheim, France). Human 143B cell line was kindly supplied by Dr F. Lecanda (CIMA, Pamplona, Spain). Cells were cultured in RPMI containing 10% fetal bovine serum at 37 °C in 5% CO₂ humidified incubators. Cell lines were routinely verified by the following tests: morphology examination, growth analysis and mycoplasma detection (MycoAlert ^TM, Lonza, Basel, Switzerland). All experiments were started with low-passaged cells (<25 times). Hypoxia (0.1% O₂, 5% CO₂, 94.5% N₂) was achieved using an InVivo₂ hypoxic workstation (Ruskinn, Bridgend, UK).

The human breast adenocarcinoma cell line KPL-4 was kindly provided by J. Kurebayashi (Kawasaki Medical School, Kurashiki, Japan). KPL-4 cells were cultured in Dulbecco's modified Eagle's medium (Sigma), supplemented with 10% FBS (Gibco) and 1% pen strep (Sigma). Cells were cultured at 37°C and 5% CO₂ and routinely passaged twice weekly until they reached the final cell concentration for injection. The cells were authenticated by the American type culture collection (ATCC) and analysed by several tests for post-freeze viability, morphology, post-freeze cell growth, interspecies determination and PCR testing of cell culture media for bacterial and fungal contamination, according to the guidelines issued by the national accreditation body for the Federal Republic of Germany (Deutsche Akkreditierungsstelle (DAkkS)). Additional mycoplasma contamination tests were performed using a commercial mycoplasma detection kit (MycoAlertTM, Lonza, Basel, Switzerland). Cells were used for implantation after 3-4 passages following thawing from frozen stocks.

Unless otherwise indicated, cell lines were obtained from DSMZ (Braunschweig, Germany) or ATCC (Molsheim, France). Culture medium, antibiotics and fetal bovine serum were from Fisher Scientific (Illkirch, France). Non-cancerous murine NIH-3T3 fibroblasts and murine MC3T3 osteoblasts were maintained in Dulbecco’s modified eagle medium (DMEM) and MEMα formulated with the addition of 10% FBS, respectively. Human T24 bladder, HeLa cervical and A549 lung cancer cells were maintained in DMEM, supplemented with 10% FBS. PC-3 human prostate cancer cells were maintained in RPMI1640, supplemented with 10% FBS. MCF7 cells stably transfected to express Fas antigen (designated as MCF7/FasR) or Fas and Bcl-xL (designated as MCF7/FasR/Bcl-xL) were donated by Dr. Vishva Dixit (Genentech Inc., San Francisco, CA, USA) and were grown in RPMI1640 containing 10% fetal bovine serum supplemented with 200 µg/mL G418 and 150 µg/mL hygromycin, as previously reported [76 (link)]. Cell lines were systematically checked by the following tests: morphological examination, growth analysis and mycoplasma detection (MycoAlert^TM, Lonza, Basel, Switzerland). All experiments were started with cells with low passages (<25 times).
TNF-α was from Bio-techne (Minneapolis, MN, USA), activating anti-Fas antibody (clone CH-11) was from Sigma-Aldrich and z-VAD-fmk was from Bachem (Bubendorf, Switzerland).