High risk clear cell renal cell carcinoma microenvironments contain protumour immunophenotypes lacking specific immune checkpoints

Abstract

Perioperative immune checkpoint inhibitor (ICI) trials for intermediate high-risk clear cell renal cell carcinoma (ccRCC) have failed to consistently demonstrate improved patient outcomes. These unsuccessful ICI trials suggest that the tumour infiltrating immunophenotypes, termed here as the immune cell types, states and their spatial location within the tumour microenvironment (TME), were unfavourable for ICI treatment. Defining the tumour infiltrating immune cells may assist with the identification of predictive immunophenotypes within the TME that are favourable for ICI treatment. To define the immunophenotypes within the ccRCC TME, fresh para-tumour (pTME, n = 2), low-grade (LG, n = 4, G1-G2) and high-grade (HG, n = 4, G3-G4) tissue samples from six patients with ccRCC presenting at a tertiary referral hospital underwent spatial transcriptomics sequencing (ST-seq). Within the generated ST-seq datasets, immune cell types and states, termed here as exhausted/pro-tumour state or non-exhausted/anti-tumour state, were identified using multiple publicly available single-cell RNA and T-cell receptor sequencing datasets as references. HG TMEs revealed abundant exhausted/pro-tumour immune cells with no consistent increase in expression of PD-1, PD-L1 and CTLA4 checkpoints and angiogenic genes. Additional HG TME immunophenotype characteristics included: pro-tumour tissue-resident monocytes with consistently increased expression of HAVCR2 and LAG3 checkpoints; an exhausted CD8⁺ T cells sub-population with stem-like progenitor gene expression; and pro-tumour tumour-associated macrophages and monocytes within the recurrent TME with the expression of TREM2. Whilst limited by a modest sample size, this study represents the largest ST-seq dataset on human ccRCC. Our study reveals that high-risk ccRCC TMEs are infiltrated by exhausted/pro-tumour immunophenotypes lacking specific checkpoint gene expression confirming that HG ccRCC TME are immunogenic but not ICI favourable.

Fig. 1. Schematic of patient characteristics and experiment workflow.

The patient characteristics (a) of the six ccRCC patients (n = 3 LG and n = 3 HG) include: patient LG_2 with a vena cava thrombus (VCT) for which we collected primary tumour microenvironment (TME) and thrombi separately but processed in the one capture array for ST-seq; patient HG_1 that we collected and processed tissues from para-TME (pTME) and TME; and patient HG_3 that we collected tissues from pTME and TME. For this experimental workflow (b), ten tissue regions were sampled from pTME, TME and VCT that excluded fibrotic and necrotic regions. ST-seq was completed using 10x Genomics Visium Gene Expression microarrayed glass slides with unique spatially barcoded ST-spots that captured the mRNA released from the overlaying thin ccRCC tissue sections. Annotation of immune ST-spots was completed with data integration of six published single-cell RNA-sequencing (scRNA-seq) datasets. Further immune cell sub-typing was completed with a scRNA and T-cell receptor (TCR) sequencing dataset. Integrated analysis was completed on CD8⁺ T cells, TAM and monocytes.

Fig. 2. Proportions of CD8⁺ T cell, TAM and monocyte sub-types within the ccRCC TME.

The proportion of immune ST-spots (a) increased with ccRCC grade. Further immune cell typing of the immune ST-spots revealed higher proportions of T cells (CD8⁺ and CD4⁺ T cells) in pTME and HG TME and macrophages in LG TME (b). Within the identified proportion of CD8⁺ T cellS, finer sub-typing (c) identified abundant non-exhausted CD8⁺ NK-like and tissue-resident cells in the pTME, LG TMEs and HG_2 TME. In contrast, exhausted CD8⁺ T cells were identified in HG_1 and HG_3 TMEs. TAM and monocyte sub-typing (d) identified non-exhausted TAM ISG^int cells within all LG and HG_2 TMEs. Abundant exhausted tissue-resident monocytes were identified in HG_1 and HG_3 TMEs. Similarly, exhausted TAM HLA^int and TAM ISG^hi were identified in pHG_1.1 and pHG_3.1. The proportion of CD8⁺ T cell (c), TAM and monocyte (d) sub-types are based on the immune cell types (b) and immune ST-spots (a) proportions. Within the HG TMEs, we further investigated heterogeneity within CD8⁺ T cells (e). For the HG TMEs, we found sub-populations within the exhausted CD8⁺ T cells expressing progenitor (TCF7) or immunomodulatory (ENTPD1) genes. Spatial mapping of the variable proportions of CD8⁺ T cells, TAM and monocytes (f) within HG ccRCC TMEs demonstrated abundant pro-tumour tissue-resident monocytes surrounding the exhausted CD8⁺ T-cell sub-types within defined tumour/immune admixed regions, as presented within the representative HG_1.2 TME.

Fig. 3. Targetable and novel IC and angiogenic gene expression within CD8⁺ T cells, TAM and monocytes.

Targetable and novel ICs (a, c) demonstrated subtle relative increased expression within CD8⁺ proliferative cells for HG_1.2, HG_1.3 and HG_3.2 TMEs. Novel IC HAVCR2 demonstrated a consistent increased expression within the exhausted tissue-resident monocytes across all TMEs. Novel IC LAG3 demonstrated increased expression within exhausted TAM ISG^hi and tissue-resident monocytes in HG_1 and HG_3 TMEs. Targetable and novel angiogenic genes (b, d) demonstrated no consistent increased expression within CD8⁺ T cells, TAM and monocytes.