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RNA Sequencing-based Transcriptome Profiling of TNBC Tumors from Mice Treated with EGFR-targetd CAR-T Therapy or Radiotherapy Alone and in Combination

ABSTRACT: We performed RNA-seq experiments on TNBC tumor tissues from mice treated with EGFR targeted CAR-T therapy or radiotherapy alone and in combination. In brief, we established E0771-EGFR TNBC cell line and inoculated the E0771-EGFR cells orthotopically into the 4th inguinal mammary fat pads of C57BL/6 mice. When the average tumor volume reached 100 mm3 on day 14, tumor bearing mice were divided to 4 groups (3 mice per group). Tumor bearing mice were given tail vein injection of 5×106 UT cells (group 1) or CAR-T cells (group 2), or exposed to 10 Gy local irradiation first then followed by tail vein injection of UT cells (group 3) or CAR-T cells (group 4). Tumor tissues were collected on day 21. Tumor tissues collected from group 1 to group 4 were named as T1, T2, T3 and T4. Total RNA from tumor was prepared using the RNeasy Plus Universal Mini Kit (QIAGEN). RNA-Seq libraries were constructed after oligo dT-based mRNA enrichment using a NEBNext mRNA Magnetic Isolation Module (New England Biolabs). The RNA-Seq Library Prep Kit (Illumina) was then used according to the manufacturer’s instructions and the cDNAs were sequenced with the Illumina Hiseq 4000.

ORGANISM(S): Mus musculus

PROVIDER: GSE168653 | GEO | 2024/03/10

REPOSITORIES: GEO

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Project description:Purpose: To compare cell states between control Her2-BBz and JUN-overexpression Her2-BBz human CAR T cells in a mouse model of osteosarcoma. Methods: 8-week old NSG mice were implanted with 1 million 143B osteosarcoma tumor cells intramuscularly in the right leg. 14 days post tumor implantation, mice were infused intravenously with 10 million human CAR+ T cells via tail vein injection. Before T cell transfer, mice were randomized for tumor size and treated with either control Her2-BBz or JUN-overexpressing Her2-BBz CAR T cells. T cells were 70% CAR+ at infusion for both groups. 14 days post T cell treatment (day 28 post tumor engraftment), 6 mice per group were euthanized, solid tumor tissue was collected, mechanically dissociated, and single cell suspensions were labeled with human-CD45 antibody and viability dye. Live hCD45+ tumor-infiltrating lymphocytes were sorted from each tumor and 6 samples were pooled for each group. ~35,000-40,000 sorted live cells (counted post sort) for each group were delivered to the Stanford Functional Genomics Facility for 3' single-cell RNA-sequencing on the 10X Genomics platform. Results: We found that substantially more JUN-overexpressing Her2-BBz CAR T cells were within the G2/M and S phases of the cell cycle than control Her2-BBz CAR T cells, consistent with increased in vivo proliferation of JUN-Her2-BBz CAR T cells. Furthermore, JUN-Her2-BBz CAR T cells also demonstrated a more activated transcriptional program (as measured by IL2RA and CD38), while many exhaustion-associated genes were downregulated (PDCD1, BTLA, TIGIT, CD200, ENTPD1, NR4A2) compared to control Her2-BBz CAR T cells. Finally, a small cluster of cells characterized by high IL7R expression (IL7R+ KLF2+ CD27+ TCF7+ SELL+) was apparent in JUN-overexpressing but not in control Her2-BBz CAR T cells, consistent with maintenance of a memory-like population capable of self-renewal. Conclusions: This study provides insights into cell states that could explain the improved efficacy of JUN-overexpression Her2-BBz CAR T cells compared to control Her2-BBz CAR T cells in a mouse model of osteosarcoma.

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RNA Sequencing-based Transcriptome Profiling of TNBC Tumors from Mice Treated with EGFR-targetd CAR-T Therapy or Radiotherapy Alone and in Combination

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