PanIN lesions are associated with a clonal bottleneck

Multiple lines of evidence suggest that PDACs arise from PanINs, which themselves arise from acinar-to-ductal metaplasia (ADM) (13, 19). We thus sought to understand how and when clonality changes during pre-malignant stages of tumor progression. To this end, we examined serial pancreatic sections from 8–10 week-old KPCX mice, a time-point at which only PanINs and ADMs were present. We found that roughly a fourth (24%) of all ADMs were polychromatic, indicating that they arose from multiple distinct acinar cells (Fig. 2A and C). By contrast, almost all PanIN lesions (97%) were found to be monochromatic (Fig. 2B and C). In the context of an ADM → PanIN → PDA model, therefore, these results suggest that premalignant progression is associated with a loss of clonal diversity during the transition from ADM to PanIN.

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Figure 2

Clonal selection occurs early in tumor progression

A and B, Fluorescent images of acinar-to-ductal metaplasia (ADM; A) and pancreatic intraepithelial neoplasia (PanIN; B) of 10 week-old KPCX mice. In A, ADMs are positive for the ductal maker Krt19 (white). Representative examples of monochromatic (top) or polychromatic (bottom) ADMs are shown. In B, representative images of monochromatic PanINs are shown.

C, Quantification of monochromatic and polychromatic ADMs and PanINs in 10 week-old KPCX mice. Data were pooled from n=3 mice, and the total number of lesions counted is shown. The bar graph on the right shows the mean percentage of ADMs and PanINs in each group. Error bars represent 95% confidence intervals. * p<0.001 by Fisher’s exact test between lesion type in ADMs and PanINs.

Scale bars 25 μm for A and 50μm for B.

Polyclonality in peritoneal and diaphragmatic metastases

We next sought to understand how clonality evolves during metastasis. To this end, we used the multifocal nature of KPCX tumors to model sub-clonal heterogeneity. In human cancers, sub-clones are defined genetically as distinct tumor populations that share a set of identical founder mutations (2, 11, 20). In this regard, each fluorescent KPCX clone shares an identical KRAS and p53 mutation but otherwise evolves independently and can thus be used to represent a distinct sub-clonal population. Using the fluorescent marker in each lesion, we then tracked their contribution to metastasis formation.

We initially examined metastases to the peritoneal wall and diaphragm. Each KPCX animal had, on average, 2–4 distinct large metastatic foci at each of these sites (Supplementary Fig. S2A, S2B, and S2C). Importantly, these metastases were spatially well-separated from the pancreas and from each other, eliminating the possibility of direct local invasion as a route of spread. Surprisingly, nearly 80% of these large lesions were polychromatic (Fig. 3A, B, and C), indicating that they originated from more than one source in the primary tumor. Likewise, examination of peritoneal and diaphragmatic micro-metastases also revealed the presence of polychromatic lesions (Fig. 3D, Supplementary Fig. S2A), demonstrating that polyclonality is present in small as well as large metastatic lesions. Interestingly, polychromatic metastases were comprised of at most two distinct populations (i.e. tri-colored metastases were not observed even if the pancreas harbored three distinctly-colored tumors). Thus, pancreatic cancer metastases to the peritoneum and diaphragm are frequently polyclonal.

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Figure 3

Polyclonal metastasis is a frequent event in murine pancreatic cancer

A, Representative fluorescent images and H&E stains of adjacent sections depicting polychromatic metastasis in the peritoneum.

B, Representative fluorescent images and H&E stains of adjacent sections depicting polychromatic metastasis in the diaphragm.

C, Total counts and percentages of metastases in each site. Data pooled from n= 7 (peritoneum) and n=9 (diaphragm) 14–16 week old tumor-bearing KPCX mice.

D, Fluorescent images of microscopic metastases from the peritoneal lining (top panel) and diaphragm (bottom panel).

Scale bars 100 μm for A and B, and 25μm for D.

Evidence that polyclonal diaphragmatic metastases come from polyclonal clusters

Polyclonal metastases could arise from the outgrowth of lesions that were polyclonal at the time of seeding or through a two-step mechanism involving seeding by one clone and subsequent recruitment (re-seeding) by another (21). Examination of ascites fluid from tumor-bearing KPCX mice revealed the presence of bi-chromatic cellular aggregates ranging in size from 2 to 50 cells in 5 out of 5 KPCX mice (Fig. 4A), suggesting that polyclonality might arise through the former mechanism.

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Figure 4

Polyclonal diaphragm metastases are seeded by polyclonal clusters

A, Bright field (i) and fluorescent images (ii) of a multi-colored cluster of disseminated tumor cells in the ascites.

B, Intraperitoneal injection of 458d_R and 458d_Y cells (30,000) either as a suspension mixture of single cells (top panel) or multi-color clusters (bottom panel) into NOD.SCID mice. Images are paired brightfield (left panel) and fluorescent (right panel) stereomicroscope images from mice 3 weeks following injection with n=4 mice for each group. Monochromatic lesions are either YFP or RFP positive and polychromatic lesions are both YFP and RFP positive.

C, Bar graph depicting mean percentage of total gross monochromatic (RFP or YFP only) or polychromatic (positive for both YFP and RFP) metastases between single cell and cluster injection groups. Data pooled from n=4 mice for each group (a total of 24 lesions were counted in the single cell group and 50 lesions were counted in the cluster group). No lesions from the single cell injection group were polychromatic. *p<0.001 by Fisher’s exact test comparing multi-color metastases between single cell and cluster injections.

D, Bar graph depicting the mean number of gross metastases in the single cell and cluster injection groups (n=4 mice in each group). Error bars represent SEM. *p=0.0026 by Student’s t-test.

Scale bars 25 μm for A and 1mm for B.

To further understand the dynamics of polyclonal metastasis formation, we performed a series of in vivo cell mixing experiments in which low passage cell lines were derived from a multi-color (RFP/YFP) diaphragmatic metastasis and then FACS sorted into their RFP (458d_R) and YFP (458d_Y) components (Supplementary Fig. S3A and S3B). We then injected 30,000 cells intraperitoneally into 6–8 week-old NOD.SCID mice, either as a 1:1 suspension of single 458d-R and 458d-Y cells or after the cells were allowed to form multi-colored clusters (Fig 4B and Supplementary Fig. S3B). After 3 weeks, diaphragmatic tissue was harvested and the number of gross monochromatic and polychromatic lesions was examined using fluorescent stereomicroscopy. Significantly, although polychromatic lesions constituted the majority (70%) of metastases following injection of cell clusters, only monochromatic lesions were seen following injection of the single cell suspension (Fig. 4B and C). Furthermore, cell clusters were more efficient than single cells at forming metastases (Fig. 4D). Taken together, these data suggest that polyclonal peritoneal metastases develop from multi-clonal aggregates shed from the primary tumor.

Polyclonality in liver and lung metastases

We then turned our attention to liver and lung, the most common sites of metastasis in human PDAC (12), which are thought to arise through hematogenous dissemination. To characterize the size distribution of metastatic foci, we counted individual lesions and binned them according to size: single cell, nano- (2–10 cells), micro- (11–100 cells), and macro-metastases (>100 cells) (Supplementary Fig. S4A and S4B). This analysis revealed that the majority of liver and lung metastases in KPCX mice were single cells or nano-metastases, with only 10–26% having more than 10 cells (Supplementary Fig. S4C). We then analyzed metastatic deposits in each size category for number and color composition. This revealed that 11–14% of nano- and micro-metastases contained two clones (Fig. 5A, B, and C); tri-colored metastases were never observed. Surprisingly, and in contrast to our findings in peritoneum and diaphragm, large polyclonal metastases were also not observed (Fig. 5B and C).

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Figure 5

Clonal evolution during metastatic growth in lung and liver

A, Representative fluorescent images of metastases in lung (top panels) and liver (bottom panels).

B, Quantification of monochromatic and polychromatic lesions in lung (top) and liver (bottom) binned according to lesion size (nano: 2–10 cells; micro: 11–100 cells; and macro: >100 cells). Percentages are relative to the total number of metastases counted for each size category. Data are pooled from n=6 (lung) and n=5 (liver) tumor-bearing animals.

C, Bar graph depicting the data presented in B. Error bars represent 95% confidence intervals. *p=0.016, **p=0.02, ***p=0.0003, and ****p=0.0016 by Fisher’s exact test.

D, Fluorescent images of bi-chromatic lung metastases depicting an increase in the ratio of the major component (CFP) relative to the minor component (RFP) as a function of lesion size.

E, Curve showing the trend depicted in (C). The ratio of the number of cells in the minor fraction relative to the total number of fluorescent cells in a metastatic lesion (y-axis) is plotted as a function of the total fluorescent cell number in the lesion (x-axis). Each (●) represents a single lung metastasis. Data were taken from 63 individual lung metastases pooled from n=6 mice (lesions with identical ratios and size are represented as a single data point). p<0.0001 by Wald Chi-square test.

Scale bars 50 μm for A and D

This result raised the possibility that the development of large metastases in the lung and liver might involve preferential outgrowth of one clone over another. To test this possibility, we quantified changes in the color composition of polychromatic liver and lung metastases as a function of cell number. Consistent with the notion of selective outgrowth, we found that lesions became increasingly dominated by a single clonal population as they increased in size (Fig. 5D and Supplementary Fig. S5A). This process was evident early, with ~90% of each polychromatic lesion dominated by one clone by the 10-cell stage (Fig. 5E and Supplementary Fig. S5B). These results indicate that while small metastatic lesions in the lung and liver may be polyclonal, there is an early drift towards monoclonality during metastatic growth in these tissues.

Cell Sorting and Culture

Pancreatic tumors were dissociated into single-cell suspensions through mechanical separation and enzymatic digestion as described (13). FACS sorting was performed using the FACSAriaII (BD biosciences) sorter at the Penn PathBioResource Flow Cytometry Core. Excitations of confetti colors were performed using the 488 nm argon laser for YFP, 405 nm violet laser for CFP, and the 532 nm red laser for RFP. Detection was performed using bandpass filters at 530/40 nm for YFP, 450/50 nm for CFP, and 575/25 nm for RFP. Cells were collected into a 2% BSA/PBS solution and cultured in pancreatic ductal cell media (13). For monitoring of fluorescent cell marker stability, >5000 cells were counted from passage 1 and passage 5 cultures following a 3–4 week interval. Total numbers of each fluorescent cell type was counted and averaged from 5 replicates.

For in vivo cell mixing experiments, cells were isolated as described above form a polychromatic diaphragmatic metastasis (mouse 458d) containing RFP and YFP populations. Cells were FACS sorted and cultured as described. No cell line authentication was performed, as all cell lines used in this paper were obtained from primary cultures of tumors derived from KPCX mice.

Image acquisition and quantification

Immunofluorescent (IF) images were acquired using both confocal and inverted fluorescent microscopes. The Zeiss LSM 710 confocal microscope employed the argon laser 488 nm line to excite nuclearGFP (nGFP), 514 nm line for YFP, 561 nm red diode laser for RFP, laserline at 458 nm for membraneCFP (mCFP), and 405 nm laser line for Dapi. Fluorescence was collected between 495–514, airy 0.41 for nGFP; 524–563, airy 0.41 for YFP; 585–622, airy 0.41 for RFP; 462–498, airy 0.41 for mCFP; and 417–494, airy 0.41 for Dapi. Images were taken with Zen 2011 software and spectral imaging coupled with image analysis using linear unmixing was performed. Imaging was performed with the Olympus IX71 inverted multicolor fluorescent microscope equipped with the following dry objective lenses: 4x (UPlanFLN NA0.13), 10x (UPlanFLN NA0.30), 20x (UPlanFLN NA0.50), 40x (UPlanFLN NA0.75). A mercury short arc lamp source (Osram) and the following excitation\emission filter sets (Chroma) were used to detect fluorescence: 535/500 nm for YFP/nGFP, 480/436 nm for CFP, 645/560 nm for RFP, 406/350 nm for Dapi, and 690/640 nm for Far Red. Images were captured with the DP71 camera (Olympus). Cytoplasmic YFP was distinguished from nuclear GFP based on cellular localization; GFP recombination events were rare compared to YFP. Histological images were captured using the Olympus BX1 light microscope attached to the DP25 camera (Olympus). Montage H&E images were taken with the EVOS FL auto cell imaging system (Thermo Fischer) with a 10x objective. Stereomicroscope images were obtained using the Lecia M216FA fluorescent microscope. Assembly and analysis of all CMYK images were performed using ImageJ 1.47v software.

Multicolor immunofluorescence and histological analysis

Tumors and tissue samples were fixed in 4% paraformaldehyde at room temperature for 30–60 minutes followed by an overnight incubation in 30% weight/volume sucrose solution. Samples were then embedded in O.C.T. (Tissue-Tek) and frozen on dry ice. Once solid, 10 μm sections were cut with a Microm HM550 cryostat (Thermo Scientific). Serial sectioning was performed on all tissue samples with 70–100 μm between sections. All sections were laid out in a similar orientation on each slide so that any given lesion could be tracked over multiple levels based on anatomical tissue landmarks and XY coordinates on the slide. Sections were stained with the nuclear marker Dapi (Life Technologies, 1:1000) and the ductal marker cytokeratin-19(24) (KRT-19, rabbit, 1:1000). For immunostaining, frozen tissues were blocked in StartingBlock (Thermo) with 2% donkey serum and 0.1% Tween-20 for 1 h, and incubated for 1h in primary antibody diluted in blocking buffer in a humidified chamber. Sections were washed three times in PBS containing 0.1% Tween-20 and incubated with DAPI and Alexa flour 633-conjugated antibodies at a 1:250 dilution for 1 h at room temperature followed by an additional 3 washes, and mounted on slides with Aqua polymount mounting reagent (Polysciences). Histological analyses were performed on adjacent frozen sections with heamtoxylin and eosin staining using standard protocols. Recombination efficiency of the Rosa^Confetti allele was examined in CX mice by counting the fluorescent cells of each color and dividing by the total Dapi positive cells. Data was gathered from 5 random pancreas fields in 5 adjacent levels. Dapi positive cells were counted using ImageJ 1.47v software resulting in >16,000 Dapi positive cells counted per pancreas. ADMs and PanINs were identified in pancreatic tissue based on typical histologic features (13) and positive staining for KRT-19.

Quantification was performed by counting the total number of ADMs and PanINs in one section from each level throughout the entirety of the pancreas. Lesions were categorized as monochromatic if >95% of cells in the lesion were of a single color and polychromatic if they contained cells of more than one color. To ensure accuracy, all PanINs that were identified as monochromatic were tracked in adjacent levels in their entirety. Analysis of metastases was performed by counting all distinct fluorescent lesions in one section from each level throughout the organ. To avoid counting larger lesions more than once, all lesions >50 cells in size were tracked in all adjacent levels in their entirety. Lung and liver metastases were binned based on the number of Dapi positive fluorescent cells in each lesion. Groupings included single (1 cell), nano (<10 cells), micro (11–100 cells), and macro (>100 cells). Lesions were scored as monochromatic if >95% of the cells in a lesion were the same color, otherwise they were considered polychromatic. Counting of lung metastases from in vivo mixing assays was performed on multiple sections taken from each organ. Diaphragmatic lesions were assessed based stereomicroscopic images. To quantitate the number of distinct lesions within each primary tumor mass, we defined a “tumor clone” as an anatomically contiguous region of monochromatic cells that shared distinct histological and IF boarders with adjacent clones as examined in multiple levels throughout the pancreatic mass.

In vivo cell mixing experimental metastasis assay

For intraperitoneal injections, equal number of cells (30,000) were injected either as a mixture of single cells or as clusters of 458d_R and 458d_Y cells into the peritoneal cavity of 6–8 week old NOD.SCID mice using an 27 gauge needle. For lung metastasis equal number of cells (20,000) were injected retro-orbitally, as previously described (22), either as a mixture of single cells or as clusters of 458d_R and 458d_Y cells into 6–8 week old NOD.SCID mice. Clusters were generated by mixing equal number of RFP and YFP cells in a low-attachment petri dish (Corning) and placed on a rocker for 8–12hrs in a 37° incubator.

We thank E. Dekleva, E. Mirek, A. Sahmoud, and C. Yang for technical assistance and N. Aiello, N. Bhagwat, D. Balli, and A. Penzo for helpful discussions. We are grateful to K. Polyak, A. Rustgi, and R. Vonderheide for comments on the manuscript, to A. Ewald for sharing unpublished data and K. Forde for assistance with statistical analysis.

Grant Support: This work was supported in part by National Institutes of Health grant CA169123 (B.Z.S.) and CA076931 (R.M.), the Penn Center for Molecular Studies in Digestive and Liver Diseases (NIH P30-DK050306), and the Abramson Family Cancer Research Institute.