A PanOmics approach towards identification and characterization of treatment resistance in B-cell lymphomas

We performed a large scale Cyclophosphamide (CTX; R-CHOP componenet) treatment based clinical trial reminiscent in vivo study to charaterize Emu-myc mouse B-cell lymphomas for their potential to relapse. Based on the findings of the experiment, the tumors which relapsed within 100 days of the observation period post-CTX were considered relapse prone (RP) and the remaining as never relapsing (NR). The primary lymphomas were then isolated from the lymph nodes of the RP or NR lymphoma bearing mice, and purified to enrich CD-19 positive cells. Using multiple OMICS-platforms,these samples were molecularly and metabolically scrutinized to account for the observed differences in drug response phenotypes i.e., NR and RP. Overall design: To categorize the Never relapsing (NR) and the Relapse prone (RP) status, individual lymphomas were propagated in up to two strain-matched, non-transgenic (i.e., genetically non-engineered wild-type), fully immuno-competent 6–8-week-old mice each via tail vein injection of 10^6 viable cells . Recipient mice were treated with a single intraperitoneal dose of cyclophosphamide (CTX, Sigma, 300 mg/kg body weight) at the time well-palpable LN enlargements (i.e., about 8–10 mm in diameter) had formed. Treatment responses were monitored during regular visits (at least twice a week; for a maximum of a 100-day observation period), which always included a general inspection of the mice and palpation of the typically affected LN regions (i.e. cervical, pre-scapular/axillar and inguinal) as time-to-relapse (TTR), i.e., progression-free survival (PFS), or reflect the time between treatment and unexpected death of the animal or a preterminal disease stage, collectively measured as overall survival (OS). After CO2 euthanasia, LN, and in some experiments also the spleen or BM, were isolated to generate tissue sections or single-cell suspensions. Lymphomas exhbiting relapse before the observation period of 100 days was considered RP and the rest as NR. These hence categorized lamphoamas were exclusively expanded in strain-matched, non-transgenic C57BL6 mice and the tumor bearing Lymph nodes were used to isolate primary lymphomas. These lymphomas were then selected for CD19-positivity using mouse B-cell isolation kit (130-090-862; Miltenyi) These cells were then used to isolate total RNA and and perform RNA-seq.