Upregulation of complement proteins in lung cancer cells mediates tumor progression

Abstract

Introduction: In vivo, cancer cells respond to signals from the tumor microenvironment resulting in changes in expression of proteins that promote tumor progression and suppress anti-tumor immunity. This study employed an orthotopic immunocompetent model of lung cancer to define pathways that are altered in cancer cells recovered from tumors compared to cells grown in culture.

Methods: Studies used four murine cell lines implanted into the lungs of syngeneic mice. Cancer cells were recovered using FACS, and transcriptional changes compared to cells grown in culture were determined by RNA-seq.

Results: Changes in interferon response, antigen presentation and cytokine signaling were observed in all tumors. In addition, we observed induction of the complement pathway. We previously demonstrated that activation of complement is critical for tumor progression in this model. Complement can play both a pro-tumorigenic role through production of anaphylatoxins, and an anti-tumorigenic role by promoting complement-mediated cell killing of cancer cells. While complement proteins are produced by the liver, expression of complement proteins by cancer cells has been described. Silencing cancer cell-specific C3 inhibited tumor growth In vivo. We hypothesized that induction of complement regulatory proteins was critical for blocking the anti-tumor effects of complement activation. Silencing complement regulatory proteins also inhibited tumor growth, with different regulatory proteins acting in a cell-specific manner.

Discussion: Based on these data we propose that localized induction of complement in cancer cells is a common feature of lung tumors that promotes tumor progression, with induction of complement regulatory proteins protecting cells from complement mediated-cell killing.

Keywords: NSCLC; RNA sequencing; complement; factor H (FH); tumor microenvironment.

Figure 1

Analysis of genes upregulated in vivo compared to in vitro cells. (A–D): Heat map of genes differentially upregulated in cells recovered from tumors (in vivo) compared to cells grown in culture (in vitro) for each of the cell lines: (A) CMT167, (B) LLC, (C) EA1, (D) EA2. Gene set enrichment analysis of Hallmark Gene Sets shows pathways upregulated in vivo compared to in vitro cells in CMT167 cells (E), LLC cells (F), EA1 cells (G), and EA2 cells (H). (I) Venn diagram shows genes commonly and/or differentially upregulated in all tumor types. (J) shows the common pathways upregulated in all 4 tumors. Differentially expressed genes were defined as being in the leading edge of the GSEA and had a rank score of less than -1, and the FPKM value had to be greater than 5.

Figure 2

Analysis of genes downregulated in vivo compared to in vitro cells. (A–D): Heatmap of genes differentially downregulated in cells recovered from tumors (in vivo) compared to cells grown in culture (in vitro) for each of the cell lines: (A) CMT167, (B) LLC, (C) EA1, (D) EA2. Gene set enrichment analysis of pathways of Hallmark Gene Sets shows pathways downregulated in vivo compared to in vitro cells in CMT167 cells (E), LLC cells (F), EA1 cells (G), and EA2 cells (H). (I) Venn diagram shows genes commonly and/or differentially upregulated in all tumor types. (J) shows the common pathways upregulated in all 4 tumors. Differentially expressed genes were defined as being in the leading edge of the GSEA and had a rank score of less than -1, and the FPKM value had to be greater than 5.

Figure 3

Complement proteins induced in vivo in murine NSCLC tumors and complement genes were found to be upregulated in vivo compared to in vitro cells. Gene Set Enrichment Analysis was performed on the data set to analyze changes in the complement pathway in vivo versus in vitro. (A) A heat map showing the upregulation of complement pathway genes in vivo. Enrichment plots for the complement pathway in CMT cells (B), LLC cells (C), EA1 cells (D), and EA2 cells (E). The “HALLMARK_COMPLEMENT” gene set from the Hallmark Gene Sets for this GSEA. The enrichment plots show the in vitro changes that positively correlate with complement genes on the left side of the plot in red while the in vivo changes negatively correlated with complement genes are on the right side in blue.

Figure 4

Role of C3 in tumor promotion FPKM values for C3 (A), C2 (B), and C4 (C) from our RNA-seq experiment show an induction of complement components C2 and C3 in vivo in 4 cell lines, with CMT and EA1 having an increase in C4 expression in vivo compared to in vitro samples. In vitro, CMT167 (D) and EA1 (E) cells were stimulated with IL1β (10 ng/mL), IFNγ (10 ng/mL), IL-22 (100 pg/mL), TNFα (10 ng/mL), IL-6 (100 pg/mL), or control (PBS) for 6, 24, 48, and 72 hours. RNA was isolated from the samples and qPCR was performed to determine what cytokines cause the induction of C3. Data were normalized to the expression of β-actin. (F) CMT167 cells were infected with C3 shRNA lentiviruses to knockdown down C3 expression. To confirm knockdown, cells were treated 10 ng/mL TNFα for 48 hours, RNA isolated, and qPCR performed to confirm knockdown in unselected, pooled samples. (G) Equal numbers of CMT-NTC or CMT-shC3-B cells were plated and proliferation was measured in the presence of TNFα over a 6 day period by quantifying cell number (H) 250K CMT-NTC or CMT-shC3-B cells were implanted into the left lung of C57Bl/6 mice, and established for 2 weeks before tumors were harvested and measured via digital calipers. Graph is combined of 2 independent experiments. N=9-11 per group per experiment. 2 outliers were removed using the ROUT test where Q=1%; a nonparametric Mann-Whitney was performed; **p<0.01.

Figure 5

Role of CD55 on tumor progression. (A) Expression of complement regulatory protein CD55 from RNA-seq data comparing in vivo and in vitro samples in all 4 cell lines, where in vivo samples appear to induce expression compared to the in vitro cells. Data presented as FPKM values. (B) Efficiency of shRNA knockdown for CD55 in CMT cells. Pools of cells transfected with either shRNA targeting CD55 or non-targeting control (NTC) were analyzed for expression of CD55 by qRT-PCR. (C) 250K CMT cells silenced for CD55 or control cells were implanted into the left lung of C57Bl/6 mice. Tumor were harvested at 2 weeks post implantation and tumor volume was assessed via digital calipers. Graph is combined of 2 independent experiments. N=7-9 per group per experiment. 2 outliers were removed using the ROUT test where Q=1%; a nonparametric Mann-Whitney was performed; ns, not significant.

Figure 6

Effect of factor H on tumor progression. (A) Factor H (Cfh) RNA Expression in vivo vs in vitro from the RNAseq data, expressed as FPKM values. In all 4 cell lines, the in vivo samples appear to induce expression compared to the in vitro cells. (B) 500K CMT cells stably transfected with factor H shRNA (shCfh-63) or control cells (NTC) were implanted into the left lung of C57Bl/6 mice, established for 2 weeks, were harvested and tumors measured. Results are the combination of 3 independent experiments where n=5-10/group/experiment. (C) 500K CMT cells with fH deleted using CRISPR (20-041) or control cells (20-039) were implanted into the left lungs of C57Bl/6 mice, established for 2.5 weeks, and tumor volume was measured using digital calipers. N=5 or 7 per group. (D) 250K EA1 cells stably transfected with factor H shRNA (shCfh-63) or control cells (NTC) were implanted into the left lung of C57Bl/6 mice, established for 2.5 weeks, were harvested and tumors measured. Results are the combination of 2 independent experiments where n=6-10/group/experiment. 1 outlier was removed using the ROUT test where Q=1%. A nonparametric Mann-Whitney test was performed on all experiments; ns, not significant; ***p<0.0001.