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Page 85 Cox et al. J Transl Genet Genom 2021;5:80-8 https://dx.doi.org/10.20517/jtgg.2021.06

Another group used single-cell RNA sequencing on brain tissue to try to determine the cause of
+
neurotoxicity. They found a very small subset of CD19 cells in the brain that also expressed CD248,
[30]
indicating that they were mural cells . While it is not yet known whether these mural cells are specifically
responsible for neurotoxicity, it is possible that they are targeted once the blood brain barrier is disrupted
during treatment. These cells are found with greater frequency in humans than mice, which highlights a
limitation of using mouse models for neurotoxicity. This makes omics studies such as these the best
modality to uncover the mechanisms that cause CART-induced toxicity in patients. This study also
highlights what can be discovered using publicly available omics data, which can often be used to ask
multiple questions.

USING OF OMICS TO INTERROGATE THE TME STATE FOLLOWING CART THERAPY
The TME is a complex system composed of a variety of cells which carry out their immunosuppressive
[31]
functions using cell-to-cell signaling, cytokines, and extracellular vesicles . These cells include regulatory T
cells, myeloid-derived suppressor cells (MDSCs), and cancer-associated fibroblasts. TME components
protect the tumor through the inhibition of effector immune cells and promotion of tumor growth. We and
others have demonstrated that inhibitory myeloid cells, cancer-associated fibroblasts, and extracellular
vesicles are capable of suppressing CART cell functions [29,31,32] . Interrogation of different components of the
TME enables the development of strategies to overcome their inhibitory effects. It has been demonstrated
that macrophages and monocytes acquire a specific transcriptomic signature in the presence of tumor cells,
and that tumor-associated macrophages undergo continuous cross talks, driven by specific regulatory genes,
such as TNFa, and CCL8 .
[33]

A myeloid cell phenotype in the TME is also associated with response in CART cell therapy. In one study,
tumor biopsies 1 month before CART cell infusion were characterized using RNA-sequencing or a
nanostring panel of 770 genes found in the TME . The group with severe neurotoxicity had significantly
[34]
lower T-cell-related genes such as CD3, CD3ζ, FOXP3, ICOS, and CD62L . The macrophage gene score
[34]
was also significantly enriched in patients who relapsed after CART cell therapy as well as increased
expression of the interferon-stimulated macrophage gene SIGLEC-1/CD169 . These data suggest that the
[35]
TME is pre-conditioned for the level of T cell infiltration and myeloid cell response that will follow CART
cell infusion.

Single-cell RNA-sequencing has been used to better understand the TME in a chemotherapy-resistant
bladder cancer patient along with a parallel study in a patient-derived xenograft model . The murine and
[36]
human results both showed upregulation of programmed death ligand 1 (PD-L1) in the remaining tumor
cells . This informed a treatment decision to use anti-PD-L1 antibody, atezolizumab, which was
[36]
[36]
successful . This study highlights how sequencing data can be utilized to inform individualized treatment
to overcome therapeutic resistance.

In metastatic melanoma, lessens are learned about tumor-infiltrating lymphocytes (TILs) in patients
receiving BRAF inhibitors (BRAFi). TCRB sequencing was performed at baseline and after 10-14 days in
patients receiving BRAFi to characterize a previously observed phenomenon that the number of TILs
[37]
increase significantly in this amount of time after treatment . They discovered that the clonality of the TILs
increased and that the majority of these were new clones. However, the patients who already had the
[37]
dominant clones prior to treatment with BRAFi achieved the best therapeutic response . Like the CART
cell study performed by Shieh et al. , this suggests that there are specific clones of T cells that expand or
[24]
survive better than others.