F7524 500ml

J774A.1 cells (ATCC number TIB-67^TM, Lot number 70040953, Manassas, VA, USA) were cultured in DMEM medium (Gibco 41965-039, UK), supplemented with penicillin (100 U/mL) and streptomycin (100 mg/mL) (Gibco 15140-122, USA), and 10% fetal bovine serum (FBS) (Sigma F7524-500ML, Mexico). Caco-2 cells were cultured in MEM-Eagle, Earle’s Salts Base (Gibco 11095080, USA), supplemented with penicillin (100 U/mL) and streptomycin (100 mg/mL), and 20% FBS. All cells were maintained in a humidified incubator with 5% CO₂ at 37 °C and routinely tested and confirmed to be free of mycoplasma contamination.

The human melanoma cell line A375 (ATCC CRL-1619) and human embryonic kidney HEK293T cells (ATCC CRL-11268) were purchased from the American Type Culture Collection (ATCC, Manassas, USA). All cells were cultured as monolayers in Dulbecco's Modified Eagle's medium (Gibco DMEM, 41965062; Thermo Fisher Scientific, Waltham, USA) with 4.5 g l⁻¹ (high) glucose supplemented with 10% fetal bovine serum (FBS, volume fraction; F7524-500 ML; Sigma-Aldrich, St Louis, USA) without antibiotics at 37°C and 5% CO₂ in a humidified atmosphere and were regularly confirmed to be mycoplasma negative. The CRISPR/Cas9 EVI/WLS knockout (KO) cell lines HEK293T KO2.9 and A375 sgEVI2_4 were generated by Oksana Voloshanenko and Iris Augustin [both Division of Signalling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Germany], respectively, using the guide RNA sgEVI2 (5′-TGGACGTTTCCCTGGCTTAC-3′) and single-cell clonal expansion, according to previously published protocols (Glaeser et al., 2018 (link)). All cell lines were authenticated.

HeLa cells are cultured in Dulbecco’s modified Eagle’s medium (11-965-092, Fisher Scientific Ltd. Canada), supplemented with 10%, fetal bovine serum (F7524-500ML, Sigma-Aldrich, USA), and 1% (volume/volume) penicillin-streptomycin (15-140-122, Thermo Fisher Scientific Inc., USA)^{29 (link)}. Cells in 2D are cultured and passaged at 80% confluence and medium change is performed every 48 hours. For the formation of 3D cancer spheroids, cells are trypsinized, resuspended to a density of $2\times {10}^6$ cell/ml, and seeded in 3% (weight/volume) agarose (16500500, ultra-pure agarose, Invitrogen, USA) microwell arrays resulting in average 267 cells per spheroid. Half medium change is done daily. At day 3, spheroids are collected and stained with $10\mathrm{\mu }\text{M}$ CellTracker Red CMTPX (C34552, Thermo Fisher Scientific Inc., USA) by 45 min incubation in a serum-free medium and washed twice with cell culture medium. Both 2D and 3D cultures are done in a humidified atmosphere with 5% carbon dioxide at 37 ${}^{\circ }$ C. For long-term storage, 3D spheroids stained with CMTPX are fixated at room temperature with 4% formaldehyde (F8775-25ML, Sigma-Aldrich, USA) for 15 min and then washed twice with Dulbecco’s phosphate-buffered saline (DPBS). For imaging experiments, the spheroids are immobilized and maintained using the developed microfluidic chip (Fig. 6a).

PDMS and OSTE round pieces were washed with 70% ethanol overnight, air dried, and washed with sterile 1× PBS overnight. Washed pieces were cultivated with 1 mL DMEM (41966-052, Thermo Fisher, Waltham, MA, USA) supplemented with 10% FBS (F7524-500 ML, Sigma-Aldrich) and 50 µL/mL Primocin (ant-pm-2, InvivoGen, San Diego, CA, USA) for 48 h in a humidified incubator at +37 °C, 5% CO₂ to mimic cell culturing environment. After 48 h, 700 µL Qiazol from miRNeasy Micro Kit (217084, Qiagen, Hilden, Germany) was directly added in wells with polymers, representing cell lysing. Wells without polymers were used as controls. Experiments were performed in duplicates. One microliter (1 × 10⁹ copies/µL) of synthetic spike-in UniSp6 from miRCURY LNA RT Kit (339340, Qiagen) was added to the lysate to monitor polymer effect on RNA isolation, cDNA synthesis, and PCR reaction since it should be similar to the control sample. cDNA was synthesized by miRCURY LNA RT Kit, and qRT-PCR was performed by miRCURY LNA SYBR Green PCR Kits (339346, Qiagen) and UniSp6 primer assay (339306, Qiagen) by applying ViiA 7 Real-Time PCR System (Thermo Fisher). Reactions were performed in technical duplicates on each biological duplicate. Ct values were compared between samples, and the p-value was calculated by the Mann–Whitney test.