Shandon cytospin

MM cells were incubated in media containing 10% FBS and Human TruStain FcX (422301; BioLegend), a Fc receptor blocking solution, for 30 min at 4°C. Cells were then washed in PBS and incubated with relevant antibodies diluted in PBS containing 3% BSA and 0.04% NaN₃ for 1 h at room temperature, followed by PBS washes. The cells were then fixed with 4% PFA and then cytospun onto the coverslip using a centrifuge (Shandon Cytospin; Thermo Fisher Scientific). Spun cells were fixed with 4% PFA followed by PBS wash. Subsequently, cells were stained with secondary antibodies conjugated with Alexa Fluor 488 or Rhodamine Red-X (Jackson ImmunoResearch) in a humidified chamber. Cells were washed with PBS and were stained with DAPI before mounting by homemade glycerol-based mounting media.

Fresh urine samples were collected and loaded into an automated cytospin machine (Shandon cytospin, Thermo Scientific, Pittsburgh, USA) following the manufacturer’s instructions and centrifuged at 1,000 revolutions per minute (rpm) for 10 min. Slides prepared by cytospin technique were prepared by alcohol-based liquid fixation and stained for Giemsa, CD10 (IR648, Dako, Glostrup, Denmark), EpCAM, WT1 (IR055, Dako, Glostrup, Denmark) and PD-L1 (ab255921, Abcam, Cambridge, UK).

Upon freeze or expansion of primary cell cultures, 5000 cells in 500 µL of culture media were transferred into a Cytofunnel and centrifuged for 5 min at 1000 RPM in a Shandon CytoSpin centrifuge (ThermoFisher cat#A78300003). Slides were dried and frozen at −70 °C. Slides were brought to room temperature submerged in 4% PFA for 15 min. Slides were washed before permeabilization with 0.02% TritonX-100 in PBS for 10 min. Blocking was done using 5% Donkey Serum in PBST for 1 h at room temperature. Primary antibody staining was done at 4 °C overnight with the following antibodies and dilutions: Vimentin 1:400 (MilliporeSigma, Burlington, MA, USA V6630), Cytokeratin 18 (R&D, Minneapolis, MN, USA AF7619), and PDGFRα/β 1:100 (abcam, Cambridge, UK ab32570). Secondary staining was done alongside DAPI using the following antibodies: Donkey anti-Mouse AlexaFluor 647 (ThermoFisher A31571), Donkey anti-Rabbit AlexaFluor 568 (ThermoFisher A10042), and Donkey anti-Sheep AlexaFluor 488 (ThermoFisher A11015). Slides were mounted in Vectashield (Vector, Burlingame, CA, USA) and imaged on a Leica DMI 6000B. Tumor cells were identified as VIM⁺CK18⁺PDGFRα/β⁻, while fibroblasts were defined as VIM⁺CK18⁻PDGFRα/β⁺. Analysis was repeated for three primary tumor lines at passages 1, 2, and 4.

Images of live cells in culture were acquired on a Zeiss Axio Observer Z1 (Carl Zeiss) microscope. For confocal imaging, cells were harvested, fixed in 2% PFA and seeded by centrifugation (800 rpm, 4 minutes) onto coated slides using a Shandon Cytospin (Thermo Fisher Scientific). After briefly washing with 1 × PBS, VECTASHIELD Antifade Mounting Medium with DAPI (Vectorlabs) was applied and slides covered with cover slips. Images were acquired on an LSM 880 with Aryscan microscope (Zeiss).