Blimp1 induces transient metastatic heterogeneity in pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic and deadly cancers. Despite the clinical significance of metastatic spread, our understanding of molecular mechanisms that drive PDAC metastatic ability remains limited. Using a novel genetically engineered mouse model of human PDAC, we uncover a transient subpopulation of cancer cells with exceptionally high metastatic ability. Global gene expression profiling and functional analyses uncovered the transcription factor Blimp1 as a key driver of PDAC metastasis. The highly metastatic PDAC subpopulation is enriched for hypoxia-induced genes and hypoxia-mediated induction of Blimp1 contributes to the regulation of a subset of hypoxia-associated gene expression programs. These findings support a model in which up-regulation of Blimp1 links microenvironmental cues to a metastatic stem cell character.

Introduction

PDAC is an almost uniformly lethal cancer that is projected to become the second leading cause of cancer-related deaths in the United States by 2030 (1). Most PDAC patients die from metastatic disease, underscoring the need to better understand the molecular mechanisms that drive disease progression and metastasis (2). Genomic analyses of PDAC have uncovered oncogenic KRAS and loss of function mutations in the CDKN2A, SMAD4, and TP53 tumor suppressors as key recurrent drivers of pancreatic cancer development (3-6). While these studies have also offered clues about metastatic progression, they have not uncovered consistent genetic alterations that explain the progression to a highly metastatic state (7-10).

While genomic alterations create stable changes that increase cancer growth, transient alterations in the metastatic state of cancer cells can be induced by interactions with stromal cells, diverse physical cues, as well as by changes in the local tumor microenvironment. For example, the epithelial-to-mesenchymal transition (EMT) is a well characterized transcriptional program that endows cancer cells with a transient high metastatic ability (11). However, EMT might not be critical for PDAC dissemination or metastasis (12,13). Subpopulations of PDAC cells with cancer stem cell-like properties have also been described but it is unclear whether these cells are the major source of metastases (14,15).

In many cancer types, metastasis is thought to be driven by diverse extracellular cues that increased stem-like behavior as well as invasion and metastasis (16). PDAC in particular has an extensive desmoplastic stromal response that generates unique physical properties, including increased extracellular matrix stiffness and areas with limited oxygen and nutrient availability (17). However, whether or not PDAC metastasis is driven by features of the tumor microenvironment is unclear. Identification of key environmental factors could provide insights into the process of metastasis as well as aid in the development of novel therapeutic strategies.

Genetically engineered mouse models of PDAC recapitulate key genetic events of the human disease. Cre-mediated expression of oncogenic Kras^G12D in pancreatic cells of loxP-Stop-loxP Kras^G12D knock-in mice (Kras^LSL-G12D/+) leads to the development of early stage pancreatic intraepithelial neoplasms (PanINs) (18). Concomitant expression of point mutant p53 or deletion of p53, Cdkn2a, and/or Smad4 allows for the development of PDAC that can progress to gain multi-organ metastatic ability (19-23). Importantly, tumors arise in vivo from genetically defined lesions and evolve in their native context, providing the opportunity to identify the cancer cell intrinsic and extrinsic processes that contribute to tumor progression.

Here, we developed a novel mouse model of human PDAC, which enabled the isolation and molecular characterization of a highly metastatic subpopulation of pancreatic cancer cells. We demonstrate that these highly metastatic cancer cells exists within hypoxic tumor areas and that the transcription factor Blimp1 drives their high metastatic potential. Gene expression signatures of the metastatic state, as well as of hypoxia-induced Blimp1-dependant genes predicts PDAC patient outcome. These findings highlight microenvironment-induced heterogeneity as a driver of pancreatic cancer progression toward its deadly metastatic phase.

Results

Generation of a system to identify and isolate a highly metastatic population of PDAC cells

The chromatin-associated protein HMGA2 is a marker of increased malignancy in many tumor types, and high HMGA2 expression predicts poor prognosis in several major human cancer types, including PDAC (24-30). To determine whether neoplastic cells in genetically engineered mouse models of human PDAC also express Hmga2, we performed immunohistochemistry (IHC) on tumors at different stages of development. Hmga2 was not expressed in cells in the normal adult pancreas or PanINs in Kras^LSL-G12D;p53^LSL-R172H/+;Pdx1-Cre (KPC) mice, but was expressed in a subset of PDAC cells (Supplementary Fig. S1A and data not shown). In human PDAC, HMGA2 expression correlates with metastasis to lymph nodes and poor prognosis, and we confirmed that high HMGA2 expression in PDAC patients predicts shorter survival (Supplementary Fig. S1B-S1D) (28,31). Together, these results document the expression of Hmga2 in a subset of cancer cells in mouse models of PDAC and confirm the correlation of the presence of cancer cells in the HMGA2^pos state with poor PDAC patient outcome.

To uncover the cellular and molecular features of Hmga2^positive and Hmga2^negative cancer cells, we generated a mouse model that would allow the isolation of these PDAC cell sub-populations. We incorporated two additional alleles into the KPC mouse model: a Cre-reporter allele (R26^LSL-Tomato) to fluorescently mark all neoplastic cells, and an Hmga2 knock-in allele, which is converted by Cre from its wild-type conformation (Hmga2^CK) into a GFP reporter (Hmga2^GFP; Fig. 1A) (20,32). In the heterozygous state (Hmga2^CK/+), the potential for GFP expression is restricted to cells in which Cre has inverted a lox-flanked region and GFP expression remains under control of all endogenous Hmga2 regulatory elements (20). In KPC;R26^LSL-Tomato/+;Hmga2^CK/+ mice (referred to as KPC^colors mice), all cancer cells were Tomato positive and Hmga2-expressing cancer cells were both Tomato and GFP positive (Supplementary Fig. S1A and S1E).

Figure 1. Identification of a subpopulation of highly metastatic pancreatic cancer cells.

(A) Alleles in the KPC^colors model (Kras^LSL-G12D/+;p53^LSL-R172H/+;Hmga2^CK/+;R26^LSL-Tom;Pdx1-Cre) before and after Cre-mediated recombination.

(B) Representative FACS plots of dissociated pancreatic cancer cells from Kras^LSL-G12D/+;p53^LSL-R172H/+;Pdx1-Cre;R26^LSL-Tom (KPCT) and KPC^colors mice. FSC/SSC-gated lineage^neg (CD45^negCD31^negF4/80^negTer119^neg) viable (DAPI^neg) Tomato^pos cells are shown.

(C) Individual primary tumors and metastases (Mets) have variable proportions of GFP^pos cells. Each dot is a tumor and the bar is the mean.

(D) Metastatic ability of GFP^neg and GFP^pos subpopulations from individual tumors was assessed by intravenous (i.v.) transplantation into recipient mice.

(E) Light and fluorescent dissecting scope images of lungs from recipient mice after i.v. transplantation of GFP^neg or GFP^pos PDAC cells from an individual tumor from a KPC^colors mouse. Scale bars = 0.5 cm.

(F) Number of cells injected and the number of metastases is indicated for each matched pair. The average number of metastases per 10⁴ GFP^neg and GFP^pos PDAC cells is shown. p-value < 0.008 by Wilcoxon matched-pair signed rank test.

The dual fluorescent marking of cancer cells in KPC^colors mice provided us with the ability to isolate Tom^posGFP^neg and Tom^posGFP^pos cancer cells by fluorescence-activated cell sorting (FACS) (Supplementary Fig. S1F-S1G). Consistent with Hmga2 expression observed by IHC, variable percentages of cancer cells in individual tumors were GFP^pos (Fig. 1B-1C). In the KPC model, progression from PanINs to adenocarcinoma is driven by loss of the wild-type p53 allele (19). Tom^posGFP^neg and Tom^posGFP^pos samples contained less than 10% remaining p53^wt allele and loss of the p53^wt allele led to the stabilization of mutant p53 protein in both GFP^neg and GFP^pos cells (Supplementary Fig. S1H-S1J). Thus, Tom^posGFP^neg and Tom^posGFP^pos cells represent two distinct sub-populations of pancreatic cancer cells.

We next performed cell culture and transplantation-based in vivo metastasis assays on GFP^neg and GFP^pos PDAC cells. GFP^pos cells consistently formed more spheres when plated into ultralow-attachment plates and formed more colonies when plated at low density under standard tissue culture conditions (Supplementary Fig. S1K and data not shown). Most importantly, for eight out of eight tumors from KPC^colors mice, the GFP^pos PDAC cells formed more metastases than their GFP^neg counterparts when transplanted intravenously into recipient mice (Fig. 1D-1F). On average, GFP^pos cells were more than 10 times more metastatic than GFP^neg cells (p-value < 0.008; Fig. 1F). Interestingly, the tumors that arose from GFP^pos cells almost always had heterogeneous GFP expression, suggesting that GFP^pos cells may be in a transient state with the potential to give rise to both GFP^pos and GFP^neg cells (Supplementary Fig. S1L-S1M).

Gene expression profiling reveals a dynamic metastatic state

To uncover pro-metastatic programs within the highly metastatic GFP^pos PDAC cell state, we performed RNA-Seq-based gene expression profiling on six pairs of GFP^neg and GFP^pos cells (Fig. 2A and Supplementary Fig. S2A-S2B). Global clustering of all samples did not clearly separate GFP^pos from GFP^neg samples (Fig. 2B). However, direct pairwise comparison of GFP^neg and GFP^pos cells uncovered hundreds of genes with consistent and significant differences (Fig. 2C-2D). Neither canonical epithelial markers nor genes related to EMT were consistently different between GFP^neg and GFP^pos cells (data not shown). We also did not observe enrichment for previously described gene expression signatures of PDAC metastasis or putative cancer stem cells in GFP^pos cells (13,14) (data not shown). Using flow cytometry, we confirmed that both GFP^neg and GFP^pos cancer cells had heterogeneous expression of the ductal/CSC marker CD133 and the epithelial marker EpCAM (Supplementary Fig. S2C-S2D) (33-35). Histologic features and IHC for differentiation markers confirmed that Hmga2 expression is largely independent from differentiated state (Fig. S2E).

Figure 2. Highly metastatic PDAC cells have a unique gene signature, which is not preserved in metastases but predicts poor patient outcome.

(A) Samples used for gene expression profiling.

(B) Consensus clustering of GFP^neg and GFP^pos PDAC cell populations using Spearman correlation.

(C) Comparison of the gene expression in GFP^neg and GFP^pos cells. Number of genes with absolute log₂ fold change > 1 and adjusted p-value < 0.05 (adjusted with maximum false discovery rate (FDR) of 0.1) is shown.

(D) Heatmap of genes differentially expressed between GFP^neg and GFP^pos PDAC cells, defined by paired comparison between GFP^neg and GFP^pos cells with a p-value < 0.05, false discovery rate < or = 0.001, and absolute log₂ fold change > 1.

(E) Heatmap of the expression of differentially expressed genes between GFP^neg and GFP^pos PDAC cells in bulk cancer cells from primary tumors and metastases from KPCT mice.

(F, G) A gene expression signature based on genes that are more highly expressed in GFP^pos cells (GFP Sig.) predicts shorter PDAC patient survival. PDAC patients from The Cancer Genome Atlas (F) and the International Cancer Genome Consortium (G) were split into top and bottom 50% (High GFP Sig. and Low GFP Sig., respectively) based on the single sample GSEA scores for GFP signature genes. p-values were calculated by log-rank test.

In addition to the paired GFP^pos and GFP^neg PDAC samples, we performed RNA-Seq analyses on FACS-sorted, bulk Tom^pos cancer cells from primary tumors and metastases (Fig. 2A and Supplementary Fig. S2F). If metastases had stable gene expression differences from primary tumors, this approach could identify gene expression alterations that contribute to metastatic ability or growth at secondary sites. Interestingly, comparison of primary tumors to all metastases identified very few significant differentially expressed genes (Supplementary Fig. S2G). Comparisons of primary tumors to liver metastases, but not to lymph node metastases, uncover several genes that were significantly differentially expressed in the liver metastases (Supplementary Fig. S2H and S2I). Consistent with a recent report on human PDAC metastasis (36), gene set analysis uncovered a trend towards enrichment for programs related to glucose metabolism in liver metastases (Supplementary Fig. S2J). Importantly, genes that were differentially expressed between GFP^pos and GFP^neg PDAC cells were not consistently different between primary tumors and metastases, consistent with the transient nature of the GFP^pos cell state (Fig. 2E). Finally, high expression of a gene signature composed of genes that were more highly expressed in metastatic GFP^pos cancer cells predicted worse PDAC patient outcome (Fig. 2F-2G).

Identification of the transcription factor Blimp1 as a driver of metastasis

To gain further insight into the metastatic process and identify potentially pro-metastatic factors, we focused on several genes that were among the most significantly and dramatically upregulated in GFP^pos cells (fold change >2; p-value <10^-6, Supplementary Fig. S3A). We stably knocked down five top candidate genes (Ero1l, Slc16a3, Glut1, Hilpda, and Blimp1) in a PDAC cell line (688M) derived from a liver metastasis from a KPCT mouse (Supplementary Fig. S3B-S3F). We assessed the importance of these genes in metastasis by quantifying the number of metastases that formed from subcutaneously and orthotopically transplanted tumors. These experiments suggested that the transcription factor Blimp1/Prdm1 could have pro-metastatic functions in PDAC (Supplementary Fig. S3G-S3L). Blimp1 is a transcription factor that was of particular interest due to its well-established role as a master regulator of cell fate determination during plasma B cell differentiation and primordial germ cell development (37,38). Blimp1 was one of the most highly upregulated genes in GFP^pos cells, being 4- to 27-fold higher in GFP^pos cells (p-value < 0.05; Fig. 3A). We confirmed increased Blimp1 protein expression in sorted GFP^pos cells relative to GFP^neg cells (Fig. 3B). Blimp1 expression was not consistently different between bulk cancer cells from primary tumors and metastases from the KPC mice, consistent with the unstable nature of the metastatic state (Supplementary Fig. S3M).

Figure 3. Highly metastatic PDAC cells express Blimp1, which is required for PDAC metastatic ability.

(A) Expression of Blimp1 (transcripts per million, TPM) in GFP^pos and GFP^neg cells (*, p-value < 0.05 by paired t test).

(B) Blimp1 protein expression in GFP^pos and GFP^neg PDAC cells from two KPC^colors tumors. Blimp1 size is estimated ∼60kD.

(C) Representative dissecting scope images of lung metastases in mice with subcutaneous tumors from 688M cells with Control or Blimp1 knockdown. Scale bars = 0.5 cm.

(D) Subcutaneous tumor growth of shControl and shBlimp1 PDAC cell line derivatives (688M). Each dot represents the average weight (g) of all tumors from a mouse and the bar is the average. In these experiments, mice were purposefully sacrificed when the subcutaneous tumors reached a designated size (Supplementary Methods). ns, not statistically significant by Student's t test.

(E) Quantification of lung metastases in mice with subcutaneous tumors. Each dot represents a mouse and the bar is the mean. Data represent pooled results from 2 experiments. **, p-value < 0.005 by Student's t test.

(F) Subcutaneous tumor growth of shControl and shBlimp1 PDAC cell line derivatives (1004M). Each dot represents the average weight (g) of all tumors from a mouse and the bar is the average. In these experiments, mice were purposefully sacrificed when the subcutaneous tumors reached a designated size (Supplementary Methods). ns, not statistically significant by Student's t test.

(G) Quantification of lung metastases in mice with subcutaneous tumors. Each dot represents a mouse and the bar is the mean. Data represent pooled results from 2 experiments. *, p-value < 0.05 by Student's t test.

(H) Representative images of lung and diaphragm metastases in KPTC;Blimp1^+/+ and KPCT;Blimp1^flox/flox mice. Scale bars = 0.5 cm. Lung and diaphragm are outlined with dotted white line.

(I) KPCT;Blimp1^flox/flox mice have fewer metastases. Representative H&E stained sections of lung and liver metastases in KPTC;Blimp1^+/+ and KPCT;Blimp1^flox/flox mice. Scale bars = 0.5 cm. Organs with Tomato^pos metastases are outlined.

(J) KPCT;Blimp1^flox/flox mice have fewer disseminated tumor cells (DTCs). Representative FACS plots of viable, lineage^negTomato^pos cancer cells in the peritoneal cavity of control KPCT;Blimp1^flox/+ and KPCT;Blimp1^flox/flox mice are shown. gated on lineage^neg single cells.

(K) Number of mice with DTCs and metastases. *, p-value < 0.05; ***, p-value < 0.0005 by Fisher's exact test. Dia, diaphragm. p-values for other comparisons between control (KPTC;Blimp1^+/+ and KPCT;Blimp1^flox/+) mice and KPCT;Blimp1^flox/flox mice are: lung, 0.1086 and Dia, 0.0502.

To further assess whether Blimp1 contributes to metastatic ability, we knocked down Blimp1 using two independent shRNAs (688M; Supplementary Fig. S3N). Blimp1 knockdown reduced the number of metastases seeded from subcutaneous tumors by >50-fold (p-value <0.005; Fig. 3C-3E). Blimp1 knockdown in a second metastasis-derived PDAC cell line (1004M) also significantly reduced metastasis (Fig. 3F-3G and Supplementary Fig. S3O). Interestingly, while Blimp1 appeared to be required for metastatic ability, overexpression of Blimp1 in multiple PDAC cell lines did not consistently enhance metastatic ability (Supplementary Fig. S3P-S3S), suggesting that expression of Blimp1 is required, but not sufficient, to drive PDAC metastasis.

Blimp1 contributes to the metastatic ability of PDAC cells in KPC mice

We next used a Blimp1 conditional knockout allele to further investigate Blimp1 function in autochthonous PDAC (37). Blimp1^f/f;Pdx1-Cre mice were viable and their pancreata did not show obvious histological changes, suggesting that Blimp1 is not required for pancreas development or homeostasis (data not shown). KPCT;Blimp1^f/f mice had similar overall pancreatic tumor burden but shorter survival compared to control KPCT;Blimp1^+/+ mice (Supplementary Fig. S3T-S3U). Pancreata from KPCT;Blimp1^f/f mice contained PanINs as well as adenocarcinomas that were similar to PDAC in control KPCT;Blimp1^+/+ mice (Supplementary Fig. S3V-S3W and data not shown). To assess the effect of Blimp1 deficiency on metastatic progression in vivo, we carefully quantified the number of Tom^pos disseminated tumor cells (DTCs) in the peritoneal cavity as well as metastases in KPCT;Blimp1^f/f and control mice. 14 out of 15 control mice (KPCT;Blimp1^+/+ and KPCT;Blimp1^f/+) developed metastases, which were often numerous and widespread in many different sites including the lymph nodes, diaphragm, lungs, and liver (Fig. 3H-3K). Conversely, only 3 out of 14 KPCT;Blimp1^f/f mice developed metastases (Fig. 3K). Additionally, peritoneal DTCs could be detected in only half of KPCT;Blimp1^f/f mice, but were present in all control mice (Fig. 3IJ-3K). Together with our observations from cell lines, these data suggest that Blimp1 promotes metastatic proclivity of PDAC.

The highly metastatic state of PDAC is associated with a strong hypoxia signature

To place Blimp1 in a pathway involved in metastasis, we next used gene set enrichment analysis (GSEA) and gene ontology (GO) enrichment analysis to identify pathways altered in the more metastatic GFP^pos cells. These analyses uncovered an overwhelming enrichment for hypoxia-induced genes in GFP^pos cells (Fig. 4A-4D and Supplementary Table S1). Genes expressed more highly in GFP^pos cells were also enriched for Hif1-binding motifs near their transcription start sites and our analyses identified significant enrichments for both Hif1 and Hif2 regulated genes in GFP^pos cells. (Fig. 4B, Supplementary Fig. S4A-S4B, and Supplementary Table S1). Conversely, genes downregulated in GFP^pos cells were enriched for cell cycle processes, consistent with hypoxia-induced cell cycle arrest (Supplementary Table S1) (39). We confirmed the up-regulation of the canonical Hif1 target gene Ero1l at the protein level in sorted GFP^pos PDAC cells (Supplementary Fig. S4C).

Figure 4. Blimp1 is regulated by hypoxia/Hif1 in murine and human pancreatic cancer cells.

(A) The GFP^pos PDAC cell state is enriched for hypoxia-induced genes and Hif-targets.

(B) Enrichment for the Hif1-binding site in the promoters of genes up-regulated in the GFP^pos cell state.

(C) Significant enrichment for hypoxia-regulated genes in GFP^pos cells. ES, enrichment score; FC, fold change.

(D) Volcano plot of the gene expression differences between GFP^pos and GFP^neg cancer cell populations. Fold change and adjusted p-values were generated by taking paired samples into consideration. Red dots denote annotated Hif1 target genes.

(E,F) Hypoxia (0.5% O₂ for 24 hours) induces Blimp1 expression in two mouse PDAC cell lines (E) and four human PDAC cell lines (F). *, p-value < 0.05; **, p-value < 0.005; ***, p-value < 0.0005 by Student's t test. Mean +/- SD of triplicate wells in shown.

(G,H) Knockdown of Hif1a in the murine 688M cells (G) and HIF1a in two human PDAC cell lines (H) reduces hypoxia-induced Blimp1 and BLIMP1 upregulation, respectively. Mean +/- SD of triplicate wells in shown. *, p-value < 0.05; **, p-value < 0.005 by Student's t test.

(I) Expression of stabilized HIF1α increases BLIMP1 expression in two human PDAC cell lines. *, p-value < 0.05; **, p-value < 0.0005 by Student's t test. Mean +/- SD of triplicate wells in shown.

(J) Chromatin immunoprecipitation (ChIP) qPCR-quantified Hif1α binding at an HRE-containing region 240 kb upstream of the Blimp1 TSS (site6). 688M cells were cultured under normoxia or hypoxia before ChIP. Six HRE were quantified for enrichment of Hif1a binding by qPCR. An HRE region in the Vegfa locus is a positive control for hypoxia-induced Hif1α binding. *, p-value < 0.05; **, p-value < 0.005 by Student's t test.

(K) The wildtype site6 (WT-HRE), but not site6 with all three HREs mutated (mut-HRE), conferred hypoxia responsiveness on a SV40 promoter or a 1.6kb Blimp1 promoter. Representative results of 688M cells transfected with indicated reporters cultured under normoxia or hypoxia. Means +/- SD of triplicated ratios of firefly luciferase normalized to co-transfected Renilla luciferase reporter are shown.

Pimonidazole-defined hypoxic areas were significantly enriched for Hmga2^pos cells (Supplementary Fig. S4D-S4E). We also employed multicolor sequential immunoflourescence staining to show that Hmga2^pos areas are enriched for the expression of the canonical hypoxic target protein Glut1 (Supplementary Fig. S4F-S4G) (40).

Based on the striking enrichment of Hif targets in GFP^pos PDAC cells from KPC^colors mice, we also determined whether Hmga2 expression is regulated by hypoxia. Under hypoxia, we noted only a slight increase in Hmga2 protein levels in PDAC cell lines (Supplementary Fig. S4H). While Hif target genes were enriched in Hmga2-expressing PDAC cells, Hmga2 knockdown had no effect on the hypoxia-induced expression of canonical Hif1 target genes (Supplementary Fig. S4I). Thus, it remains unclear why Hmga2 marks this highly metastatic PDAC subpopulation but these data suggest that other aspects of the in vivo microenvironment either in conjunction with, or independent from, hypoxia induce Hmga2 expression in these cells.

Blimp1 is a novel hypoxia/Hif-regulated gene in human and murine PDAC

To determine whether Blimp1 expression is regulated by hypoxia in human and murine PDAC, we assessed Blimp1 mRNA and protein expression in PDAC cell lines exposed to hypoxia (0.5% O₂ for 24 hours). Hypoxia led to the induction of multiple canonical Hif target genes, Hif1α stabilization, and the prominent and consistent induction of Blimp1 in two mouse and four human PDAC cell lines (Supplementary Fig. S4J-S4K and Fig. 4E-4F). Hypoxia-mediated induction of Blimp1 in mouse and human PDAC cells was attenuated by Hif1α knockdown, suggesting that Hif1α is at least partially required for Blimp1 induction under these conditions (Fig. 4G-4H). BLIMP1 induction in human PDAC cell lines was also partially HIF2 dependent (Supplementary Fig. S4B). Expression of stable HIF1α was sufficient to increase BLIMP1 expression in PDAC cells (Fig. 4I). Finally, human PDACs with the highest BLIMP1 expression are enriched for hypoxia signatures relative to those with the lowest BLIMP expression (Supplementary Fig. S4L and data not shown).

We next investigated how hypoxia and Hif induce Blimp1 expression. To determine whether Blimp1 can be induced indirectly by secreted factors, we measured Blimp1 levels in PDAC cells cultured with conditioned media from hypoxia-treated cells or recombinant VEGF-A which has been shown to induce Blimp1 in endothelial cells (41). In both cases, we did not observe robust Blimp1 induction (Supplementary Fig. S5A-S5C). Blimp1 was induced rapidly after exposure to hypoxia, paralleling the kinetics of canonical Hif target genes, suggesting that Blimp1 might be induced directly by Hif (Supplementary Fig. S5D). Analysis of chromatin accessibility around the Blimp1 locus (see below) enabled the prioritization of multiple putative distal regulatory regions that contained hypoxia-response elements (HREs) (Supplementary Fig. S5E). Hif1a ChIP qPCR identified a cluster of three adjacent HREs upstream of Blimp1 that were bound by endogenous Hif1α in PDAC cells under hypoxia (Fig. 4J). This HRE-containing putative distal regulatory region conferred hypoxia responsiveness in a heterologous reporter system, which was abolished by mutation of its HRE motifs (Fig. 4K and Supplementary Fig. S5F-S5G). Furthermore, Blimp1 knockdown significantly reduced the ability PDAC cells cultured under hypoxia to form spheres and had a variable effect of migratory ability in cell culture. These results suggest a role for Blimp1 in cellular behaviors related to metastatic ability (Supplementary Fig. S6A-S6I).

Blimp1 regulates a subset of hypoxic-mediated gene expression changes in PDAC

To characterize Blimp1's function in hypoxic cells, we profiled the gene expression and genome-wide chromatin accessibility of shControl and shBlimp1 PDAC cells cultured under normoxic and hypoxic conditions (Fig. 5A). Hypoxia can induce changes in chromatin state, and Blimp1 has been implicated in both plasma cell precursors and primordial germ cells as a regulator of chromatin structure (42-44). We uncovered widespread hypoxia-induced changes in chromatin accessibility, with differentially accessible regions being enriched for Hif-binding elements (Fig. 5B and Supplementary Fig. S6J-S6K). In addition, hypoxia induced genes associated with newly open chromatin regions more than those with constitutively open or close regions, suggesting that hypoxia likely regulate target gene induction in part through chromatin accessibility changes (Supplementary Fig. S6L). Interestingly, Blimp1 knockdown had minimal impact on hypoxia-induced changes in chromatin accessibility, indicating that the function of Blimp1 is largely independent of its ability to recruit factors that lead to changes in chromatin state (Supplementary Fig. S6M-S6N).

Figure 5. Blimp1 regulates the expression of a subset of hypoxia-induced genes.

(A) The PDAC cell line 688M was cultured for 24 hours under normoxia/hypoxia in vitro for RNA-Seq and ATAC-Seq analyses.

(B) Pearson correlation of all samples based on global chromatin accessibility determined from ATAC-Seq analysis.

(C) Clustering of correlation of samples based on the global gene expression derived from RNA-Seq analysis.

(D) Hypoxia-induced changes in gene expression. Numbers of differentially expressed genes that are significant with FDR < 0.001 are shown.

(E) Genes suppressed by hypoxia are significantly enriched for cell cycle-related programs (E2F_TARGETS). Genes induced by hypoxia are significantly enriched for a hypoxia signature. ES, enrichment score; FC, fold change.

(F,G) Gene expression differences between shBlimp1 and shControl cells cultured under hypoxia (F) and normoxia (G). Numbers of differentially expressed genes that are significant with FDR < 0.001 are shown.

(H) Blimp1-repressed genes under hypoxia are significantly enriched for cell cycle-related programs (bottom panel). GSEA was conducted by comparing the transcriptomes of shControl and shBlimp1 cells cultured under hypoxia. ES, enrichment score; FC, fold change.

(I) Blimp1 knockdown reduces the induction of hypoxia-induced genes (blue) while de-represses hypoxia-suppressed genes (red). Change of ratio under Blimp1 knockdown is defined as the ratio of a, “fold change of gene expression induced by hypoxia in shBlimp1 cells” over b, “fold change of gene expression induced by hypoxia in shControl cells”. p-values are adjusted with a FDR = 0.1.

(J) About 35% of hypoxia-induced genes required Blimp1 for their full induction and were less induced by hypoxia in shBlimp1 cells (log₂ fold change < -0.5).

(K) GO term analysis of hypoxia-induced, Blimp1-dependent genes defined in (J). Significantly enriched biological processes are shown. Node size represents odds ratio (log₁₀) and color shows adjusted p-values. Thickness of lines connecting nodes represents percent of shared genes between connected processes. Hypoxia (bold) and cell mobility (black font) related processes are highlighted.

(L) Hypoxia-induced, Blimp1-dependent genes (BLIMP1 Sig.) predict PDAC patient outcome. Single sample GSEA (ssGSEA) scores for the BLIMP1 Sig. were used to separate the top three from the bottom quartile of TCGA patients.

Our parallel RNA-Seq analysis identified many genes that were dramatically and significantly altered by hypoxia (Fig. 5C-5D). As expected, canonical genes related to hypoxia were induced while cell cycle-related programs were suppressed (Fig. 5E). Consistent with the induction of Blimp1 by hypoxia, Blimp1 knockdown affected the expression of more genes when the cells were cultured under hypoxic conditions (Fig. 5C, Supplementary Fig. S6O, and comparison between Fig. 5F and 5G). Blimp1 was required for both the induction and repression of subsets of hypoxia-regulated genes (Supplementary Table S2). Under hypoxia, cell cycle-related programs were enriched in shBlimp1 cells compared to shControl cells, suggesting that Blimp1 might play a role in hypoxia-induced cell cycle arrest (Fig. 5H). Approximately 12% of hypoxia-repressed genes required Blimp1 for their full suppression (N=95 of 825 hypoxia-repressed genes; Fig. 5I and Supplementary Fig. S6P-S6Q). The majority of these hypoxia-repressed, Blimp1-dependent genes were related to cell cycle processes, consistent with the role of Blimp1 in suppressing proliferation during plasma B cell differentiation (Supplementary Fig. S6R) (45,46).

Additionally, approximately 35% of hypoxia-induced genes required Blimp1 for their full induction and were less induced under hypoxia in shBlimp1 cells (N=833 of 2342 hypoxia-induced genes; Fig. 5J). Genes encoding proteins involved in hypoxic responses and cell mobility were reduced in shBlimp1 cells compared to shControl cells (Fig. 5K, Supplementary Fig. S7A-S7B, and Supplementary Table S3). We found that accessible distal regulatory regions within 500 kb of the transcription start sites of Blimp1-dependent, hypoxia-induced genes were enriched for transcription factor-binding motifs that closely resemble the Blimp1 motif (47) (IRF1/IRF2, Supplementary Fig. S7C-S7D). Although the regulation of these Blimp1-dependant genes is likely to be multifaceted, the enrichment of these motifs suggests that at least a subset of these genes may be regulated directly by Blimp1. Finally, high expression of a gene expression signature composed of hypoxia-induced, BLIMP1-dependent genes predicted worse PDAC patient outcome (Fig. 5L). These results suggest that Blimp1 is a hypoxia-regulated gene that regulates a defined subset of hypoxia-controlled genes in PDAC cells.

Blimp1 is required for hypoxia-induced cell cycle repression and the induction of pro-metastatic genes

To gain additional insight into the function of Blimp1 in PDAC, we integrated our ex vivo RNA-Seq data from GFP^neg and GFP^pos PDAC cells with our in vitro RNA-Seq data from shControl and shBlimp1 cells cultured under normoxia and hypoxia. As anticipated, a vast majority of genes that are more highly expressed in GFP^pos cells were also up regulated by hypoxia in PDAC cells in cell culture (Fig. 6A and Supplementary Fig. S8A-S8B). Furthermore, many hypoxia-induced genes that were more highly expressed in GFP^pos cells in vivo required Blimp1 for optimal induction under hypoxic conditions in vitro (Supplementary Fig. S8A and S8C). These results underscore the strong hypoxia signature in the GFP^pos cells and highlight the contribution of Blimp1 to the expression of these genes.

Figure 6. Blimp1 regulates the expression of pro-metastatic, hypoxia-induced target genes.

(A) Genes that are significantly differentially expressed under hypoxia (absolute log₂ fold change > 1 and FDR < 0.001) are differentially expressed between GFP^pos and GFP^neg cells (Fisher's exact test, quadrant counts shown, p-values < 0.0001).

(B) BLIMP1 expression correlates with a subset of hypoxia-induced genes in human PDAC. All genes from the TCGA PDAC dataset are ranked by their correlation with BLIMP1 expression (Pearson r) and enrichments of 36 BLIMP1-dependent (red) and 36 BLIMP1-independent (grey), hypoxia-induced genes are shown. NS, enrichment score; NES, normalized enrichment score; pval, nominal p-value.

(C-E) Blimp1 deficiency in KPCT mice significantly increases PDAC cell proliferation (C) while reduces Car9 (D) and Glut1 (E) expression (N = 3). Proliferation was measured by IHC for phospho-histone 3 (H3P). H3P positive nuclei were quantified. (C) Each dot is the number of H3P^pos cells in a field and the bar is the mean. (D,E) Each dot is the sum of all pixels (brown color) above the cutoff in a field and the bar is the mean. *, p-value < 0.05; ***, p-value < 0.0005 by Student's t test.

(F) Expression (TPM) of Hilpda in GFP^pos and GFP^neg PDAC cells from KPC^colors mice. Paired samples are connected with a line. *, p-value < 0.05 by paired t test.

(G) Blimp1 knockdown significantly reduced hypoxia-induced Hilpda expression. **, p-value < 0.005. ns, not statistically significant.

(H-J) Hilpda is required for PDAC metastasis from subcutaneous tumors. (H) Each dot represents the mean of multiple subcutaneous tumor weight in a mouse and the bar is the mean of all mice in each cohort. (I) Each dot represents the number of lung metastases in a mouse and the bar is the mean. (J) Representative fluorescent images of lung metastases from the subcutaneous tumors are shown. Lung lobes are outlined with dotted lines (J). ns, not statistically significant; *, p-value < 0.05 by Student's t test. Scale bars = 0.5 cm.

(L) Proposed model. Dotted lines indicate potentially indirect mechanisms.

To further relate these gene expression programs with BLIMP1 expression in human PDAC, we used these integrated datasets to define a 36-gene signature of Blimp1-dependent, hypoxia-induced genes that are also higher in the GFP^pos state. Across multiple human PDAC gene expression datasets, this Blimp1 signature correlated with BLIMP1 expression, suggesting conserved mechanism of BLIMP1 function in human PDAC in vivo (Fig. 6B and Supplementary Fig. S8D-S8E).

Our gene expression profiling suggested that Blimp1 might be required for hypoxia-induced cell cycle arrest. To directly test this, we cultured shControl and shBlimp1 cells at 0.5% and 20% O₂ and assessed proliferation by short term BrdU labeling. While shControl cells almost completely arrested under hypoxia, shBlimp1 cells continued to proliferate (Supplementary Fig. S8F-S8G). To determine whether Blimp1 reduces the proliferation of PDAC cells in tumors in vivo, we assessed the proliferation of cancer cells in pancreatic tumors in KPCT;Blimp1^f/f and control KPCT;Blimp1^+/+ mice. Cancer cells in autochthonous Blimp1-deficient tumors had a higher mitotic index (Fig. 6C and Supplementary Fig. S8H). The higher proliferation of cancer cells in tumors from KPCT;Blimp1^f/f mice is also consistent with the shorter survival of KPCT;Blimp1^f/f mice (Supplementary Fig. S3U).

Many of the genes that were hypoxia-induced, Blimp1-dependent, and expressed at higher levels in the more metastatic GFP^pos PDAC cells have been previously implicated as pro-metastatic factors in other cancer types. These genes included Pgf, Dusp1, Hmox1, Car9, Glut1, and Hilpda (48-53). Consistent with our RNA-Seq data, we observed reduced Glut1 and Car9 protein expression in PDACs in KPCT;Blimp1^f/f mice compared to KPCT mice (Fig. 6D-6E and Supplementary Fig. S8H-S8M). High expression of the lipid droplet-associated protein Hilpda in other cancer types correlates with disease progression and metastasis (53,54). Hildpa expression was higher in GFP^pos PDAC cells, induced by hypoxia in murine and human PDAC cells, and its induction was partially Blimp1-dependent (Fig. 6F-6G and Supplementary Fig. S4J). Hilpda knockdown reduced metastasis in our initial analysis and we further confirmed that Hilpda knockdown in PDAC cells significantly reduced their metastatic ability (Fig. 6H-6J, Supplementary Fig. S8N, and Supplementary Fig. S5G-S5L). These data suggest that Hilpda is a Blimp1-regulated pro-metastatic factor in PDAC.

Discussion

To uncover the molecular mechanisms that drive the metastatic ability of PDAC, we initially took two unbiased gene expression-profiling approaches: analysis of Hmga2-GFP^pos and Hmga2-GFP^neg PDAC subpopulation as well as analysis of bulk cancer cells from large primary tumors and macro-metastases. In both cases, we specifically isolated cancer cells at high purity by FACS to avoid confounding our analyses with contaminating stromal cell populations. Analysis of bulk cancer cells from primary tumors and metastases uncovered few significant gene expression changes, implying that cancer cells in the largest primary tumors possess most of the molecular features required for metastatic spread.

These findings are in stark contrast to the extensive gene expression differences between large primary tumors and metastases that we uncovered in a parallel study on a Kras^G12D-driven, p53-deficient mouse model of lung adenocarcinoma (55). In the lung cancer model, large primary tumors often existed in an earlier non-metastatic state that had profound gene expression differences from metastases. In the lung, oncogenic Kras^G12D alone can drive extensive tumor growth and even tumors in Kras^LSL-G12D;p53^flox/flox mice do not immediate receive benefit from being p53 deficient (55-58). Thus, pancreatic tumors may be forced into a potentially metastatic state by the selective pressures of primary tumor growth, thereby explaining the high likelihood of metastatic spread even in patients with relatively small pancreatic tumors (59).

Despite these observations, multiple lines of evidence suggest that the metastatic ability of PDAC is still an acquired phenotype. We previously noted mice with widespread PanIN lesions that lack any disseminated tumor cells in the peritoneal cavity (60). Additionally, we and others have generated mice with clonally marked pancreatic tumors and documented that not all tumors give rise to metastases (60,61). Although we did not observe gene expression differences between large primary tumors and metastases, we have documented microenvironment-driven metastatic heterogeneity. Our results support a model in which the development of hypoxic regions generates cells with increased metastatic ability (62). Consistent with results from autochthonous mouse models, human PDAC is a highly hypoxic cancer type (63) and the metastatic ability of orthotopically-grown, patient-derived PDAC xenografts is predicted by their level of hypoxia (64).

Hypoxia has been shown to induce metastasis in multiple cancer types through various mechanisms (reviewed in 64, 67, and 68). For example, hypoxia is related to alterations in EMT/MET, angiogenesis, local invasion and intravasation, extravasation, as well as the formation of the pre-metastatic niche (65). While some consequences of hypoxia may be relatively generalizable across cancer types, some outputs of hypoxia may also be cancer type specific, thus the importance of Blimp1 in these different steps of the metastatic cascades as well as in different cancer types remain to be determined.

Hypoxia also has a tremendous impact on the self-renewal and differentiation of the progenitor/stem cell lineages. For example, hypoxia potentiates the engraftment of human hematopoietic stem cells in recipient mice (66,67) and also helps maintain the stemness of embryonic stem and iPS cells in culture (68,69). In several cancer types, hypoxia has also been shown to play important roles in maintaining cancer stem cells (CSCs). In brain tumors, hypoxia promotes and/or maintains cancer cell stemness similar to the effect of hypoxia on bona fide stem cells (70,71). Several studies have identified subpopulations of murine and human PDAC cells with CSC characteristics based on their ability to generate new tumors upon transplantation (14,72). Importantly, the highly metastatic PDAC state that we identified is not directly related to previously reported CSC populations, the differentiation state of the cancer cells, or EMT. Thus, whether the highly metastatic PDAC cell state and these CSC states represent parallel or partially overlapping programs will be an important area for future study.

We initially anticipated that the high metastatic ability of Hmga2-expressing cells would be driven by Hmga2 itself. Surprisingly, this is not the case, as Hmga2 deficiency has no impact on the metastatic ability of tumors in the KPC PDAC mouse model nor does it influence the induction of canonical hypoxia target genes (BMG, S-HC, MMW; manuscript in preparation and Supplementary Fig. S4I). Hmga2 could play a subtle role in the later stages of metastatic outgrowth or may simply be a marker of the metastatic state.

Mechanistically, our results uncover hypoxia-mediated induction of the transcription factor Blimp1 as one molecular link between the tumor microenvironment and transient induction of pro-metastatic gene expression programs in PDAC. Although our data show that Blimp1 can be induced through hypoxia-mediated stabilization of Hif, other factors within the tumor microenvironment may also impact Hif activity (Fig. 6L). In PDAC, Blimp1 functions as a molecular switch that promotes metastatic ability while suppressing cell division under hypoxia (Fig. 6L). Our results are consistent with the link between Blimp1 and migratory ability of human lung and breast cancer cell lines in vitro (73,74). Blimp1 has not been described as a hypoxia/Hif target gene in normal cell types but hypoxia may also influence Blimp1 expression in those settings. In early embryos, where oxygen levels are low prior to the formation of major blood vessels (75), Blimp1 is expressed in primordial germ cells where it represses somatic programs and helps maintain pluripotency (38,76). Blimp1 is also critical for the differentiation of plasma cells that are generated in secondary lymphoid organs and maintained in bone marrow, both of which have hypoxic regions (37,77,78).

In summary, our findings support the concept of microenvironmental, rather than mutational, evolution being a critical factor that fosters PDAC metastatic ability. We found that intratumoral hypoxia, which is an inevitable feature of advanced human PDAC, induces the expression of the pro-metastatic transcription factor Blimp1. The co-option of this master regulatory transcription factor is required for metastatic ability, and the molecular output of Blimp1 expression is the modulation of discrete hypoxia-induced gene expression programs. A greater understanding of the origins and molecular features of cancer cells with transient high metastatic ability could provide therapeutic opportunities to reduce metastatic spread and further our appreciation of the obligate plasticity of these cells during the metastatic process.

Methods

Mice

Kras^LSL-G12D, p53^LSL-R172R, Blimp1^flox, Pdx1-Cre, Rosa26^LSL-tdTomato, and Hmga2^CK mice have been described (18,20,32,37,79,80). Mice with the Kras^LSL-G12D and the R26^LSL-tdTomato alleles in cis on chromosome 6 were used to maximize retention of the R26^LSL-tdTomato allele even in genomically unstable tumors. 6-10-week-old NOD/Scid/γc (NSG) mice (The Jackson Laboratory, Stock number: 005557) were used for transplantation experiments. The Stanford Institutional Animal Care and Use Committees approved all animal studies and procedures.

Histology and quantification of IHC

All histological staining were performed on paraffin-embedded, formalin-fixed sections as described previously (60). Briefly, 4 μm sections were re-hydrated and subjected to antigen retrieval before IHC was performed using Vector Lab ABC Vectastain kit. We used custom FIJI macro scripts for the quantification of IHC. See Supplemental Experimental Procedures for the detail of staining procedures and IHC quantification.

RNA-Seq data analyses

RNA and genomic DNA samples were extracted from 10⁴ to 5×10⁴ sorted cancer cells using Qiagen AllPrep DNA/RNA Micro Kit. RNA from ex vivo FACS-purified cells (15 ng/sample) were converted to cDNA and amplified with the NuGEN Ovation RNA-Seq system. Subsequently, amplified cDNA was sonicated and subjected to library preparation using the Illumina TruSeq DNA sample preparation kit. Total RNA from shControl or shBlimp1 688M cells cultured in 0.5% or 20% O₂ were used for the preparation of RNA-Seq libraries with Illumina's TruSeq RNA Library Prep Kit v2 according to manufacturer's protocol. Sequencing was performed on Illumina HiSeq 2000 for 100-bp paired-end (ex vivo samples) and non-paired-end (in vitro samples) reads. See Supplemental Experimental Procedures for RNA-Seq analysis details.

ATAC-Seq data analysis

Murine PDAC 688M cells cultured in 0.5% or 20% O₂ were also used for ATAC-Seq library preparation. Briefly, nuclei were extracted before incubation with TDE1 Tn5 transposase (Illumina). The fragmented genomic DNA was PCR amplified and ATAC-Seq libraries were sequenced on an Illumina NextSeq with paired-end 76bp reads using an Illumina High Output kit. ATAC-Seq data were processed as previously described with some modifications (81). See Supplemental Experimental Procedures for ATAC-Seq analysis details.

Western blotting

Cell lysates were prepared with RIPA buffer supplemented with protease inhibitors. Proteins were separated by PAGE before being transferred onto a Bio-Rad PVDF membrane. Primary antibodies were incubated in the presence of 5% skimmed milk at 4°C overnight, followed by stringent washes (3×) before staining with horseradish-peroxidase-conjugated secondary antibodies. After additional washes, enhanced chemiluminescence was performed to visualize the proteins of interest. See Supplemental Experimental Procedures for more detail of western blot analyses.

Hypoxia induction and qRT-PCR

To induce hypoxia in vitro, cancer cells were seeded at sub-confluency and cultured in a hypoxia chamber (Invivo2-400, Ruskin Technologies) with 0.5% O₂ for 24 hours before harvest. Cells were subsequently lysed with TRIzol (Thermo Fisher Scientific, 15596-018) directly on tissue culture dishes for RNA extraction. RNA concentration was quantified on a NanoDrop spectrophotometer (Thermo Fisher Scientific, NanoDrop 2000 UV-Vis Spectrophotometer) and converted to cDNA according to manufacturer's protocol (Thermo Fisher Scientific, 4368814). For the quantification of transcripts, SYBR green (Sigma-Aldrich, S9194) was used with specific primer pairs. β-actin was used as internal control. See Supplementary Data for more detailed information.

Cell lines

None of the cell lines used in this study were authenticated. The years when the PDAC cell lines were obtained are as follow: 688M, 2014; 1004M, 2014; 887M, 2017; 1814, 2015; 1810, 2015; human PDAC cell Panc1, Colo357, BxPC1, AsPC1, and Capan1 were all obtained in 2014. All PDAC cell lines that were used in experiments reported in this study were expanded early in the passage and aliquots were stored in liquid nitrogen. Thawed cells were passaged within 1-2 months for any experimental usage.

Subcutaneous transplantation of cell lines into NSG mice

688M and 1004M PDAC cells were cultured at sub-confluency shortly before harvest for transplantation. All cells used in the transplantation experiments were validated for knockdown efficacies of designated genes. Briefly, cells were trypsinized and washed 3× in cold PBS before subcutaneous injection. 2.5 × 10⁵ cells were used per injection on the dorsal flank. The numbers of Tom^pos metastases in the lung were quantified by direct counting using a fluorescence dissecting scope. Alternatively, H&E sections were used to estimate lung metastases seeded by the Tomato-negative 1004M cells. Metastases in the lung were validated by histology.

Pancreatic orthotopic transplantation

688M PDAC cell derivatives validated for efficient knockdown were washed 3× in cold PBS before resuspension in 100% Matrigel (Corning, 356231). Subsequently, a surgical procedure was performed with direct injection of the cells/Matrigel mixture into the pancreas of NSG mice. See Supplemental Experimental Procedures for more detail of the orthotopic transplantation.

Statistical analysis

For comparison between two quantitative variables, we used Student's t test when samples were not paired and paired t test for paired samples. When more than two variables were compared, both one-way ANOVA and Kruskall-Wallis test were used. For comparison of survival in Kaplan-Meier analyses, we used log-rank test for univariate survival analyses. Fisher's exact test was used in the analysis of contingency tables. Analyses were performed using Prism 6.0 (Graphpad Software Inc.).

Significance.

PDAC is an almost uniformly lethal cancer, largely due to its tendency for metastasis. We define a highly metastatic subpopulation of cancer cells, uncover a key transcriptional regulator of metastatic ability, and define hypoxia as an important factor within the tumor microenvironment that increases the metastatic proclivity.