multi tissue cdna panel  (TaKaRa)

Journal: Cancer Discovery

Article Title: TCR-Engineered T Cells Directed against Ropporin-1 Constitute a Safe and Effective Treatment for Triple-Negative Breast Cancer

doi: 10.1158/2159-8290.CD-24-0168

Figure Lengend Snippet: ROPN1/B is absent in healthy tissues and highly and homogenously present in primary and metastatic TNBC independent of pretreatment. A, Flowchart of the discovery of ROPN1/B as a target for T-cell treatment of TNBC and validation of its tumor-restricted expression using healthy tissues, as well as primary and metastatic TNBC tissues. Intracellular targets (CGAs: n = 276) were screened for absent expression in healthy tissues ( n = 1,479 tissues) and expression in TNBC ( n = 191 tissues). ROPN1/B mRNA and protein expression was further validated in multiple sets of healthy tissues (two additional cohorts) and four different cohorts of patient tumor tissues (see “Methods” for details). B, Dot plot shows gene expressions as fold changes relative to GAPDH (2 −dCt ) according to RT-qPCR using a cDNA library of 48 healthy tissue samples. NY-ESO1 was used as a reference. Green, ROPN1 ; purple, ROPN1B ; gray, NY-ESO1 ( CTAG1B ), n = 2 to 3 per target antigen. C, Representative immune stainings of ROPN1/B using an array of 14 healthy tissues (2–6 donors per tissue, total n = 66). D, Violin plots show the distribution of gene expression of NY-ESO1 ( CTAG1B , gray) and ROPN1 (green) in TNBC cohort 1 ( n = 66, geTMM normalized) and cohort 2 ( n = 183, fRMA-normalized; see “Methods” for details on cohorts). Data were analyzed using the Wilcoxon signed-rank test: cohort 1: P < 2.2E16; cohort 2: P < 2.2E16. E, Representative immune stainings of TNBC categorized according to staining intensity with different intensities for NY-ESO1 (top) and ROPN1/B (bottom). Stacked bar graphs (middle) show the fraction of TNBC tumors with weak, moderate, and strong immune staining of NY-ESO1 or ROPN1/B [tissue microarrays (TMA), n = 311]. Stacked bar graphs (right) show fractions of TNBC tumors with either 1% to 9%, 10% to 25%, 26% to 50%, or 51% to 100% of tumors cells positive for NY-ESO1 or ROPN1/B protein; the latter category of 51% to 100% ROPN1-stained cells is further subdivided into the fractions: 51% to 75%, 76% to 90%, and 91% to 100% (in zoomed-in stacked bar). F, Violin plot shows the distribution of gene expressions of ROPN1/B in primary and metastatic TNBC from TNBC cohort 1 ( n = 66, geTMM normalized) and cohort 3 ( n = 22) following batch correction. Data were analyzed using the Wilcoxon signed-rank test: P = 0.61. G, Stacked bar graphs (left) show the fraction of primary and metastatic TNBC with weak, moderate, and strong immune staining of ROPN1/B (whole tissue sections, n = 15 paired samples). Stacked bar graphs (right) show the fraction of primary and metastatic TNBC with either 1% to 9%, 10% to 25%, 26% to 50%, or 51% to 100% of tumor cells positive for ROPN1/B. H, Violin plots show the distribution of gene expression of ROPN1/B in pre- and post-induction treatment biopsies of metastatic TNBC retrieved from cohort 4 ( n = 53 of which n = 44 are paired samples, geTMM normalized). Data of paired pre/postsamples was analyzed using the Wilcoxon signed-rank test: cisplatin: n = 8, P = 0.4; cyclophosphamide: n = 10, P = 1; doxorubicin: n = 9, P = 0.4; irradiation: n = 7, P = 0.7; no induction: n = 10, P = 0.73. ACT: L. intestine, Large intestine; S. intestine, small intestine.

Article Snippet: Quantitative PCR (qPCR) was performed on cDNA panels of 48 healthy human tissues (OriGene Cat# HMRT104, Technologies, Rockville, MD) and TNBC cell lines using MX3000 [RRID:SCR_020526; Taqman probes ROPN1 : Hs00250195_m1; ROPN1B : Hs00250195_m1; CTAG1B (NY-ESO1): Hs00265824_m1; GAPDH : Hs02758991_g1; NBL1 : Hs01063631_m1; UBE2O : Hs00222904_m1; FAAH: Hs01038664_m1].

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, cDNA Library Assay, Gene Expression, Staining, Irradiation