Multiregional single-cell proteogenomic analysis of ccRCC reveals cytokine drivers of intratumor spatial heterogeneity

Miheecheva, Natalia; Postovalova, Ekaterina; Lyu, Yang; Ramachandran, Akshaya; Bagaev, Alexander; Svekolkin, Viktor; Galkin, Ilia; Zyrin, Vladimir; Maximov, Vladislav; Lozinsky, Yaroslav; Isaev, Sergey; Ovcharov, Pavel; Shamsutdinova, Diana; Cheng, Emily H.; Nomie, Krystle; Brown, Jessica H.; Tsiper, Maria; Ataullakhanov, Ravshan; Fowler, Nathan; Hsieh, James J.

doi:10.1016/j.celrep.2022.111180

Cited by 15 publications

(8 citation statements)

References 73 publications

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“…We show that Cx3cl1-expressing tumor cells drive enrichment of immunosuppressive CD206 Hi macrophages, and depletion of inflammatory monocytes and neutrophils. This finding is in line with previous reports of chemokine-driven spatial organization of immune cells within tumors 23,24 . However, antibody depletion of Cx3cl1 was insufficient to potentiate the anti-PD-1 blockade + anti-CD40 agonist combination in CIT9-RFP immunosuppressive tumors (data not shown), suggesting that additional mediators of immunosuppression contribute to the "coldness" of these tumors.…”

Section: Discussionsupporting

confidence: 93%

Tumor cell heterogeneity drives spatial organization of the intratumoral immune response in squamous cell skin carcinoma

Tanaka

Lum

et al. 2023

Preprint

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Intratumoral heterogeneity (ITH)—defined as genetic and cellular diversity within a tumor—is linked to a failure of immunotherapy in multiple cancer types and to an inferior anti-tumor immune response even in the absence of therapy. To determine how tumor heterogeneity shapes the immune microenvironment and impacts responses to therapy, we modeled heterogeneous tumors comprised of a pro-inflammatory ("hot") and an immunosuppressive ("cold") tumor population. This was done by mixing two squamous skin carcinoma cell lines that alone give rise to tumors with a reproducible immunologically "hot" or "cold" immune phenotype respectively, and labeling these with YFP or RFP fluorescent tags to enable precise spatial tracking. The resulting mixed-population tumors were made up of a patchwork of distinct regions that could be classified as being comprised of YFP+ ("hot") cells, RFP+ ("cold") cells, or a mixture of YFP+ and RFP+ cells. We find the spatial organization of tumor cells creates a blueprint for the spatial organization and functional activity of infiltrating immune cells, defining the architecture of both tumor-infiltrating T cell and myeloid cell compartments. Using a combination of microdissection techniques and single-cell spatial transcriptomics, we show that YFP regions harbor a higher frequency of Th1 cells and IFNγ+ CD8 T cells compared to RFP regions, whereas immunosuppressive macrophages preferentially accumulate in RFP regions. Total CD4 T cells were also enriched in YFP regions, but by contrast, total CD8 T cells—despite their superior function in YFP regions—were of low abundance throughout the heterogeneous tumors. We identify the chemokine Cx3cl1, produced at higher levels by our "cold" tumors, as a mediator of intratumoral macrophage abundance, and show that overexpression of Cx3cl1 in pro-inflammatory ("hot") tumor cells leads to an increase in immunosuppressive CD206+ macrophages. We further interrogated the impact of this spatial patterning of immune cells on the responses of heterogeneous tumors to checkpoint blockade. We find that combination of PD-1 blockade and CD40 agonist is able to increase the Th1 response in "cold" RFP regions, however the T cell response in "cold" regions remains inferior to "hot" regions and treatment achieves only a modest reduction in tumor growth. Collectively, our results demonstrate that the spatial organization of heterogeneous tumor cells has a profound impact on directing the spatial organization and function of tumor-infiltrating immune cells as well as on responses to immunotherapy.

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Section: Discussionsupporting

confidence: 93%

Tumor cell heterogeneity drives spatial organization of the intratumoral immune response in squamous cell skin carcinoma

Tanaka

Lum

et al. 2023

Preprint

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“…In silico modeling of ccRCC has suggested that tumor growth limited to the tumor periphery may be associated with higher subclonal diversification and enhanced genomic ITH [27]. Remarkably, an association between genomic ITH and composition of the tumor microenvironment that was mediated by tumor-derived cytokines has been reported [28]. The latter finding lends further support to the idea of co-evolution between tumor cells and the microenvironment.…”

Section: Discussionmentioning

confidence: 65%

Digital Spatial Profiling Identifies the Tumor Periphery as a Highly Active Biological Niche in Clear Cell Renal Cell Carcinoma

Schneider,

Kaczorowski,

Jurcic

et al. 2023

Cancers

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Clear cell renal cell carcinoma (ccRCC) is characterized by a high degree of intratumoral heterogeneity (ITH). Besides genomic ITH, there is considerable functional ITH, which encompasses spatial niches with distinct proliferative and signaling activities. The full extent of functional spatial heterogeneity in ccRCC is incompletely understood. In the present study, a total of 17 ccRCC tissue specimens from different sites (primary tumor, n = 11; local recurrence, n = 1; distant metastasis, n = 5) were analyzed using digital spatial profiling (DSP) of protein expression. A total of 128 regions of interest from the tumor periphery and tumor center were analyzed for the expression of 46 proteins, comprising three major signaling pathways as well as immune cell markers. Results were correlated to clinico-pathological variables. The differential expression of granzyme B was validated using conventional immunohistochemistry and was correlated to the cancer-specific patient survival. We found that a total of 37 proteins were differentially expressed between the tumor periphery and tumor center. Thirty-five of the proteins were upregulated in the tumor periphery compared to the center. These included proteins involved in cell proliferation, MAPK and PI3K/AKT signaling, apoptosis regulation, epithelial-to-mesenchymal transition, as well as immune cell markers. Among the most significantly upregulated proteins in the tumor periphery was granzyme B. Granzyme B upregulation in the tumor periphery correlated with a significantly reduced cancer-specific patient survival. In conclusion, this study highlights the unique cellular contexture of the tumor periphery in ccRCC. The correlation between granzyme B upregulation in the tumor periphery and patient survival suggests local selection pressure for aggressive tumor growth and disease progression. Our results underscore the potential of spatial biology for biomarker discovery in ccRCC and cancer in general.

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“…Through numerous signaling pathways and networks, proteins can promote cell proliferation and migration, contributing to tumorigenesis and cancer progression [ 25 , 26 ]. A cell’s proteome is a collection of functional molecules, and the central dogma dictates how genetic information flows between the genome, epigenome, and proteome.…”

Section: Discussionmentioning

confidence: 99%

Screening and Identification of a Prognostic Model of Ovarian Cancer by Combination of Transcriptomic and Proteomic Data

et al. 2023

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The integration of transcriptome and proteome analysis can lead to the discovery of a myriad of biological insights into ovarian cancer. Proteome, clinical, and transcriptome data about ovarian cancer were downloaded from TCGA’s database. A LASSO–Cox regression was used to uncover prognostic-related proteins and develop a new protein prognostic signature for patients with ovarian cancer to predict their prognosis. Patients were brought together in subgroups using a consensus clustering analysis of prognostic-related proteins. To further investigate the role of proteins and protein-coding genes in ovarian cancer, additional analyses were performed using multiple online databases (HPA, Sangerbox, TIMER, cBioPortal, TISCH, and CancerSEA). The final resulting prognosis factors consisted of seven protective factors (P38MAPK, RAB11, FOXO3A, AR, BETACATENIN, Sox2, and IGFRb) and two risk factors (AKT_pS473 and ERCC5), which can be used to construct a prognosis-related protein model. A significant difference in overall survival (OS), disease-free interval (DFI), disease-specific survival (DSS), and progression-free interval (PFI) curves were found in the training, testing, and whole sets when analyzing the protein-based risk score (p < 0.05). We also illustrated a wide range of functions, immune checkpoints, and tumor-infiltrating immune cells in prognosis-related protein signatures. Additionally, the protein-coding genes were significantly correlated with each other. EMTAB8107 and GSE154600 single-cell data revealed that the genes were highly expressed. Furthermore, the genes were related to tumor functional states (angiogenesis, invasion, and quiescence). We reported and validated a survivability prediction model for ovarian cancer based on prognostic-related protein signatures. A strong correlation was found between the signatures, tumor-infiltrating immune cells, and immune checkpoints. The protein-coding genes were highly expressed in single-cell RNA and bulk RNA sequencing, correlating with both each other and tumor functional states.

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Multiregional single-cell proteogenomic analysis of ccRCC reveals cytokine drivers of intratumor spatial heterogeneity

Cited by 15 publications

References 73 publications

Tumor cell heterogeneity drives spatial organization of the intratumoral immune response in squamous cell skin carcinoma

Tumor cell heterogeneity drives spatial organization of the intratumoral immune response in squamous cell skin carcinoma

Digital Spatial Profiling Identifies the Tumor Periphery as a Highly Active Biological Niche in Clear Cell Renal Cell Carcinoma

Screening and Identification of a Prognostic Model of Ovarian Cancer by Combination of Transcriptomic and Proteomic Data

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