Histologic variants in bladder cancer share aggressive molecular features including a CA125+ cell state and targetable TM4SF1 expression

Histologic variant (HV) subtypes of bladder cancer are clinically aggressive tumors that are more resistant to standard therapy compared to conventional urothelial carcinoma (UC). Little is known about the transcriptional programs that account for the morphological and biological differences in HV tumors. To investigate the tumor biology of HV bladder cancers, we generated a single cell RNA sequencing (scRNA-seq) atlas of nine HV tumors and three UC tumors. Our analyses revealed a tumor cell state specific to HVs that is characterized by expression of MUC16 (CA125), KRT24, and WISP2. This CA125+ cell state bears transcriptional hallmarks of epithelial-mesenchymal transition, is enriched in metastases, is predicted to be highly chemotherapy resistant, and is linked with poor survival, suggesting that this cell state plays an important role in the aggressive biology of HV tumors. Our analyses also provide evidence of transcriptional “mimicry” between HVs and histologically similar non-urothelial cell types. Lastly, we identified higher expression of TM4SF1, a cell surface protein associated with cancer metastasis, in HV tumor cells compared to UC tumor cells. Chimeric antigen receptor (CAR) T cells engineered against TM4SF1 protein demonstrated in vitro and in vivo activity against bladder cancer cell lines in a TM4SF1 expression-dependent manner, highlighting its potential as a therapeutic target in bladder cancer. cDNA library preparation was performed using the Seq-Well platform as previously described. Briefly, 10,000-20,000 cells were loaded onto a Seq-Well array containing 110,000 barcoded mRNA capture beads (ChemGenes, Ct: MACOSKO-2011-10(V+)). Arrays were sealed using a polycarbonate membrane (Sterlitech, Cat: PCT00162X22100) at 37 °C for 40 minutes. Cells were then lysed in lysis buffer (5 M guanidine thiocyanate, 1 mM EDTA, 0.5% sarkosyl, 1% BME) for 20 minutes at room temperature. Hybridization of mRNA to the beads was performed in hybridization buffer (2 M NaCL, 4% PEG8000) for 40 minutes. The beads were then collected and washed with 2 M NaCl, 3 mM MgCl2, 20 mM Tris-HCl pH 8.0, 4% PEG8000. Reverse transcription was then performed using Maxima H Minus Reverse Transcriptase (ThermoFisher, Cat: EP0753) in Maxima RT buffer, PEG8000, template switch Oligo dNTPs (NEB, Cat: No447L), and RNase inhibitor (Life Technologies, Cat: AM2696) at room temperature for 15 minutes and then 52 °C overnight. Second strand synthesis was performed using Klenow Exo- (NEB, Cat: M0212L) in Maxima RT buffer, PEG8000, dNTPs, and dN-SMRT oligo for 1 hr at 37 °C. Whole transcriptome amplification was performed with KAPA HiFi Hotstart Readymix PCR kit (Kapa Biosystems, Cat: KK2602) and SMART PCR Primer. The reactions were purified using SPRI beads (Beckman Coulter) at 0.6X and then 0.8X volumetric ratio. Libraries were prepared using 800-1000 pg of DNA and the Nextera DNA Library Preparation Kit. Dual-indexing was performed using N700 and N500 oligonucleotides. Library products were purified using SPRI beads at 0.6X and then 1X volumetric ratio. A final 3 nM dilution was prepared for each library and sequenced on an Illumina NovaSeq S4 flow cell.