Abstract
We have demonstrated that orthotopic tumor (intramuscular injection of the human rhabdomyosarcoma, RMS, cell line RH30 in NSG mice) can be treated with FGFR4-specific human CAR-T cells in the context of reversing the immunosuppressive activity of myeloid cells. CAR-T cells invoke a robust stromal response featuring the presence of M2 macrophages, and the deposition of collagen. The countermeasures employed were: deletion of MIF in RH30, ATRA to differentiate MDSC, anti-PD1, inhibition of TGFβ with SD208, IDO1 with epacadostat, iNOS with L-NAME, and CSF-1 with pexidartinib. Together, these interventions promoted effective CAR-T therapy. To better understand the biology of the CAR-T treated tumors, we analyzed the myeloid cell types present using digital spatial profiling (DSP, GeoMx, Nanostring, Inc.). Regions of tumor from control untreated tumor (TO), tumor treated with untransduced activated T-cells (UTD), and tumor treated with FGFR4-specific CAR-T were stained with F4/80, regions captured, then analyzed for gene expression. Regions of interest (ROI) within tumors included F4/80 negative "tumor-center” regions, and F4/80 positive regions that were either muscle proximal or tumor associated. PCA analysis of GeoMx gene sets revealed distinct gene expression patterns according to the treatment administered (TO, UTD, or FGFR4 CAR-T), proximity to normal muscle tissue, or localization within tumor.
Gene set enrichment analysis (GSEA) for TO-Myeloid ROI identified skeletal muscle FAP cell and fibroblast-like cell gene expression sets, even though this was an F4/80 positive region. Muscle proximal myeloid cells from UTD-treated tumor also identified skeletal muscle FAP, and tissue resident macrophage (TRM) signatures. Tumor associated myeloid cells from UTD-treated tumor expressed skeletal muscle T cell and myeloid expression patterns, as well as those for TRM. Tumor associated myeloid cells in CAR-T treated tumor identified a differentiating stem cell gene expression data set in addition to TRM and skeletal muscle myeloid cell gene expression patterns. Analysis of individual transcripts driving GSEA scores revealed upregulation of collagen transcripts in T cell treated tumors, in agreement with our histochemical studies. Immune associated genes like CCL2 were also enriched in UTD and CAR-T treated tumor-associated myeloid regions, indicating a positive feedback loop for further monocytic infiltration. The enriched transcript values for F4/80 (Adgre1) CSF1R, CD206, CD169, and TREM2 confirm our DSP F4/80 selection. We found strong upregulation of B2M in UTD and CAR-T treated myeloid cells, indicative of IFN-γ signaling. The lack of IFN-γ transcripts in the myeloid cell populations themselves, implies CAR-T cells are the source. Identifying transcripts of known function by gene expression analysis of DSP ROIs support also supports the relevance of the unique transcripts identified.
The induction of Gas6 in muscle proximal myeloid cells implies a specific anatomical localization for this pro-tumorigenic population. Ireland L et al., 2020, reported blockade of Gas6 reverses tumor EMT and supports NK activation in a pancreatic adenocarcinoma model. Dai et al., 2022, reported that Gas6 polarizes decidual macrophages to an M2 phenotype promoting maternal immunotolerance, highlighting its relevance in the stromal rich environment induced by CAR-T therapy. Lymphocyte antigen 6 complex, locus E (Ly6E, TSA-1, SCA-2) a prognostic marker in colorectal carcinoma, and the MS4A family (which includes CD20) member MS4A6c were also identified as markers of tumor-associated myeloid cells. The origin of myeloid cells within an i.m. tumor lesion can arise from mis-differentiated bone marrow-derived cells that infiltrate the lesion, from monocytes responding to tissue-generated signals, from tissue resident macrophages already present, or from innate myeloid cells present in muscle tissue that were generated embryonically from liver or even earlier from the AGM (aorta gonad mesonephros), and which have their own intrinsic tissue regulatory biology. The strongest transcript expressed was for an uncharacterized protein, GM52800, indicating there is much to discover in the biology of tumor-associated myeloid cells, especially in the context of CAR-T therapy.
Disclosures
Orentas:Miltenyi Biotec: Research Funding.
