Identification and editing of stem-like cells in methylcholanthrene-induced sarcomas

Abstract

The cancer stem cell (CSC) paradigm posits that specific cells within a tumor, so-called CSC-like cells, have differing levels of tumorigenicity and chemoresistance. Original studies of CSCs identified them in human cancers and utilized mouse xenograft models to define the cancer initiating properties of these cells, thereby hampering the understanding of how immunity could affect CSCs. Indeed, few studies have characterized CSCs in the context of cancer immunoediting, and it is currently not clear how immunity could impact on the levels or stem-like behavior of CSCs. Using the well-studied 3'methylcholanthrene (MCA) model of primary sarcoma formation, we have defined a CSC-like population within MCA-induced sarcomas as expressing high levels of stem cell antigen-1 (Sca-1) and low levels of CD90. These Sca-1⁺CD90^- CSC-like cells had higher tumor initiating ability, could spontaneously give rise to Sca-1-negative cells, and formed more sarcospheres than corresponding non-CSC-like cells. Moreover, when examining MCA-induced sarcomas that were in the equilibrium phase of cancer growth, higher levels of CSC-like cells were found compared to MCA-induced sarcomas in the escape phase of cancer progression. Notably, CSC-like cells also emerged during escape from anti-PD-1 or anti-CTLA4 therapy, thus suggesting that CSC-like cells could evade immune therapy. Finally, we demonstrate that paradoxically, interferon (IFN)-γ produced in vivo by immune cells could promote the emergence of CSC-like cells. Our findings define the existence of a Sca1⁺CD90^- CSC-like population in the MCA-sarcoma model capable of differentiation, tumorsphere formation, and increased tumor initiation in vivo. These cells may also act as mediators of immune resistance during cancer immunoediting and immune therapy.

Keywords: MCA Sarcoma; cancer immune surveillance; cancer immunoediting; cancer stem cells; immune therapy.

Figure 1.

Identification of CSC-like cells in murine MCA-sarcoma cell lines. Analysis of the CSC-like potential of the various subpopulations based on Sca-1 and CD90 expression in the MCA-sarcoma cell lines F244 and F535. F244 was sorted based on Sca-1 expression into Sca-1⁺ and Sca-1⁻ populations. F535 was sorted into Sca-1⁺CD90⁻ and Sca-1⁺CD90⁺ populations. Each subpopulation was analyzed for their capacity to: (A) reconstitute the initial cell line heterogeneity in vitro by cultivating the sorted subpopulations and analyzing the reconstitution of the initial cell line heterogeneity over time by FACS analysis of cell surface expression of Sca-1 and CD90, (B) form sarcosphere in anchorage and serum-independent conditions, shown are the number of spheres per number of cells seeded ± S.E.M. (n = 3 in each condition), and (C) initiate tumor formation in vivo in Rag2^−/− × γc^−/−. Survival curves show the percentage of tumor-free mice over time after transplantation of each subpopulation. Histograms present the percent of tumor-bearing mice at the end of the experiment. The presented data are combining two independent experiments. *reflects the p values obtained in between groups at a given time point calculated by the Student t test, while the indicated p values represent the significance between the two groups in the global experiment calculated by the log-rank (Mantel-Cox) test. All the experiments were repeated at least twice.

Figure 2.

Primary and transplanted MCA sarcomas in equilibrium are enriched in CSC-like cells. (A) Transplanted F244 or 4862 MCA sarcoma cell lines or primary MCA-sarcoma tumors undergoing equilibrium or progressive growth were harvested and the percentage of CSC-like cells (Sca-1⁺CD90⁻) as a percentage of total tumor cells (CD45⁻CD44⁺CD29⁺) was assessed by FACS ex vivo and shown as a bar graph or (B) representative dot plots. (C) Sarcosphere-forming capacity of cell lines originating from equilibrium or representative progressive tumor transplants. Mean sarcosphere number ± S.E.M. is shown. Left panel shows the cell line 4862 and right panel shows the cell line F244. The experiment was repeated at least twice. (D) Cancer initiating property of cell lines derived from equilibrium tumors is greater than that of cell lines derived from progressing tumors. Cell lines were transplanted into in Rag2^−/− × γc^−/− mice at the indicated number of cells and the percentage of mice developing tumors at endpoint is shown.

Figure 3.

Therapeutically-induced immune responses enriched the CSC-like population of MCA-sarcoma. (A) The cell line F244 was transplanted into syngeneic WT animals treated with either anti-CTLA4 (orange line), anti-PD-1 (blue line) or corresponding isotype controls (black line). Escaping tumors, defined by a tumor progression delayed by the checkpoint blockade treatment (anti-CTLA4, n = 2; anti-PD-1, n = 3) compared to the isotype control treatment (n = 8), are highlighted on the tumor kinetic graph (Left panel). (B-C) Tumors from part (A) were harvested at endpoint, and CSC percentages were analyzed immediately ex vivo without adapting to cell culture. (B) Scatter plot and (C) representative FACS plots are shown. The experiment was repeated at least three times.

Figure 4.

Emergence of CSC-like cells in vivo required IFN-γ production. (A) The cell line F244 was transplanted into syngeneic Rag1^−/− (n = 6) or WT (n = 10) mice and CSC-like cells (Sca-1⁺CD90⁻) as a percentage of total tumor cells (CD45⁻CD44⁺CD29⁺) were assessed by FACS ex vivo. (B) The cell line F244 was transplanted into syngeneic WT mice treated with blocking anti-IFN-γ antibodies (n = 10) or isotype controls (n = 4). CSC-like cells were measured as in part (A). (C) Sarcosphere-forming capacity of cell lines originating from F244 tumor transplants in either Rag1^−/− or WT recipients. Mean sarcosphere number ± S.E.M. is indicated (n = 3). (D) The cell line F244 was treated or not with 100U/ml of IFN-γ. Cell surface expression of Sca-1 and CD90 was analyzed at 30 minutes, 1, 2, 4, 6, 8 and 24 hours. (E) The cell line F244 was treated or not with 100U/ml of IFN-γ. Cells were washed and subsequently treated or not with 100U/ml of IFN-γ for 7 days and cell surface expression of Sca-1 and MHC Class I was analyzed All experiments in this figure were repeated at least twice.