FAP Delineates Heterogeneous and Functionally Divergent Stromal Cells in Immune-Excluded Breast Tumors

Cremasco, Viviana; Astarita, Jillian L.; Grauel, Angelo L.; Keerthivasan, Shilpa; MacIsaac, Kenzie D.; Woodruff, Matthew C.; Wu, Michael; Spel, Lotte; Santoro, Stephen P.; Amoozgar, Zohreh; Laszewski, Tyler; Migoni, Sara Cruz; Knoblich, Konstantin; Fletcher, Anne; LaFleur, Martin W.; Wucherpfennig, Kai W.; Puré, Ellen; Dranoff, Glenn; Carroll, Michael; Turley, Shannon J.

doi:10.1158/2326-6066.cir-18-0098

Cited by 143 publications

(134 citation statements)

References 63 publications

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“…This was reflected in our data, in which expression of multiple pericyte markers was observed in the CAF3 population as well as in PDPN+ lymph node FRCs. A similar phenomenon was reported in a subcutaneous model of breast cancer, in which expression of pericyte markers was also observed in FRC populations 41 . Thus, the most robust method to differentiate between these cell types is to assess whether they are associated with endothelial vessels.…”

Section: Discussionsupporting

confidence: 81%

Single-cell RNA sequencing reveals a dynamic stromal niche within the evolving tumour microenvironment

Davidson

Efremova

Riedel

et al. 2018

Preprint

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Non-cancerous stromal cells represent a highly diverse compartment of the tumour, yet their role across tumour evolution remains unclear. We employed single-cell RNA sequencing to determine stromal adaptations in murine melanoma at different points of tumour development. Naive lymphocytes recruited from lymph nodes underwent activation and clonal expansion within the tumour, prior to PD1 and Lag3 expression, while tumourassociated myeloid cells promoted the formation of a suppressive niche through cytokine secretion and inhibitory T cell interactions. We identified three temporally distinct cancerassociated fibroblast (CAF) populations displaying unique signatures, and verified these in human datasets. In early tumours, immune CXCL12/CSF1 and complement -expressing CAFs supported recruitment of macrophages, whereas contractile CAFs became more prevalent in later tumours. This study highlights the complex interplay and increasing diversity among cells that co-evolve with the tumour, indicating that from early stages of development, stromal cells acquire the capacity to modulate the immune landscape towards suppression.In particular, extensive heterogeneity has been reported within tumour fibroblast populations.Cancer associated fibroblasts (CAFs) are the most abundant non-immune stromal component, secreting growth factors, promoting angiogenesis, facilitating metastasis and regulating immune infiltrates 9-15 . Although they express typical fibroblast markers, such as fibroblast activation protein (FAP), platelet derived growth factor receptors α (PDGFRα) and β (PDGFRβ), podoplanin (PDPN), Thy-1 and α-smooth muscle actin (αSMA), no single marker universally identifies all CAFs within the tumour stroma 16-18 . To date, many studies rely on positive selection approaches, in which one or two markers are used to isolate CAFs for functional characterisation. Consequently, these findings likely reflect a sub-population of cells and may bias our perceptions of CAF function. It remains unclear whether fibroblast subpopulations with distinct roles are present in the tumour microenvironment.Current approaches lack the resolution to visualise the true extent of stromal heterogeneity and may mask rare populations, or cellular phenotypes, that could be critical for tumour survival. Therefore, we have employed single-cell RNA sequencing (scRNAseq) to profile both immune and non-immune stromal populations from the B16-F10 murine model of melanoma. Furthermore, cells were isolated from both primary tumours and draining lymph nodes, at different stages of tumour development, enabling a systems level interrogation of the melanoma microenvironment in real-time. Here, we identified the presence of a diverse immune landscape, in which the composition and phenotype of leukocytes change as the tumour evolves. In particular, effector T cells displaying signs of dysfunction, were detected predominantly in late stage tumours. This work also highlighted significant heterogeneity within the CAF compartment of the primary tumour. Three distinc...

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Section: Discussionsupporting

confidence: 81%

Single-cell RNA sequencing reveals a dynamic stromal niche within the evolving tumour microenvironment

Davidson

Efremova

Riedel

et al. 2018

Preprint

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“…Since the initial report on different subpopulations of αSMA+ myofibroblasts in breast cancer (Lazard et al, 1993), it is now becoming clear that CAFs come with different flavours and that a heterogeneous population exists within a single breast tumour (Bartoschek et al, 2018;Busch et al, 2017;Calvo et al, 2013;Costa et al, 2018;Cremasco et al, 2018;Raz et al, 2018;Su et al, 2018). Here, we have presented data showing the existence of numerous CAF subpopulations in murine models of TNBC, expanding on previous findings within breast cancer that have proposed the existence of only 2 to 4 different CAF subpopulations (Bartoschek et al, 2018;Costa et al, 2018;Cremasco et al, 2018;Raz et al, 2018).…”

Section: Co-existence Of Numerous Caf Subpopulations In Breast Cancermentioning

confidence: 67%

“…Intra-tumoural heterogeneity of cancer cells in breast cancer is well recognised, and emerging evidence points toward breast cancer CAFs being equally heterogeneous (Bartoschek et al, 2018;Busch et al, 2017;Calvo et al, 2013;Costa et al, 2018;Cremasco et al, 2018;Raz et al, 2018;Su et al, 2018). While cancer cell heterogeneity is believed to arise through clonal evolution, the origins of CAFs, and how this influences the CAF heterogeneity, are still being intensely studied.…”

Section: Introductionmentioning

confidence: 99%

Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer

Venning

Zornhagen

Wullkopf

et al. 2020

Preprint

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Cancer-associated fibroblasts (CAFs) comprise a heterogeneous population of stromal cells within the tumour microenvironment. CAFs exhibit both tumour-promoting and tumour-suppressing functions, making them exciting targets for improving cancer treatments. A careful identification and characterisation of the CAF heterogeneity is thus necessary before implementing CAFtargeted strategies in cancer. With that in mind, we developed a flow cytometry strategy based on exclusion of non-CAF cells and successfully employed it to explore the temporal heterogeneity of CAFs in two models of triple-negative breast cancer (4T1 and 4T07). Analysing 128 murine tumours we identified 5-6 main CAF subpopulations and numerous minor ones based on the analysis of alpha smooth muscle actin, fibroblast activation protein alpha, platelet derived growth factor receptor alpha and beta, CD26/DPP4 and podoplanin. All markers showed temporal changes, and CD26+ CAFs emerged as a large novel subpopulation. These results form the foundation needed for the future elucidation of tumour-promoting CAF subpopulations.

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“…In addition to malignant cells, the alteration of the stroma of solid tumors, namely the activation of cancer-associated fibroblasts has been described as critical determinant of cancer development and survival, and was also confirmed as therapeutic target [2]. It was shown that the expression of FAP prolyl endopeptidase, even on the cell surface or solubilized in the plasma, correlates with the malignancy and cisplatin resistance of carcinomas [29,[44][45][46]. Therefore, we have developed an assay to measure the activity of FAP cleaving the Fmoc-Gly-Pro-Cysteic acid-Ile-Gly-NH 2 peptide substrate ( Figure S2).…”

Section: Discussionmentioning

confidence: 97%

Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

Balog

Hackler²,

Kovács³

et al. 2019

IJMS

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The treatment of metastatic breast cancer remained a challenge despite the recent breakthrough in the immunotherapy regimens. Here, we addressed the multidimensional immunophenotyping of 4T1 metastatic breast cancer by the state-of-the-art single cell mass cytometry (CyTOF). We determined the dose and time dependent cytotoxicity of cisplatin on 4T1 cells by the xCelligence real-time electronic sensing assay. Cisplatin treatment reduced tumor growth, number of lung metastasis, and the splenomegaly of 4T1 tumor bearing mice. We showed that cisplatin inhibited the tumor stroma formation, the polarization of carcinoma-associated fibroblasts by the diminished proteolytic activity of fibroblast activating protein. The CyTOF analysis revealed the emergence of CD11b+/Gr-1+/CD44+ or CD11b+/Gr-1+/IL-17A+ myeloid-derived suppressor cells (MDSCs) and the absence of B220+ or CD62L+ B-cells, the CD62L+/CD4+ and CD62L+/CD8+ T-cells in the spleen of advanced cancer. We could show the immunomodulatory effect of cisplatin via the suppression of splenic MDSCs and via the promotion of peripheral IFN-γ+ myeloid cells. Our data could support the use of low dose chemotherapy with cisplatin as an immunomodulatory agent for metastatic triple negative breast cancer.

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FAP Delineates Heterogeneous and Functionally Divergent Stromal Cells in Immune-Excluded Breast Tumors

Cited by 143 publications

References 63 publications

Single-cell RNA sequencing reveals a dynamic stromal niche within the evolving tumour microenvironment

Single-cell RNA sequencing reveals a dynamic stromal niche within the evolving tumour microenvironment

Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer

Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

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