Case selection and primary tumors
Because of the need to fully understand the pathogenesis of IPMNs and their derivative invasive cancers, we attempted in vivo and in vitro propagation of these lesions. We initially attempted to grow IPMNs with and without an invasive component, but after initial success with in vivo propagation, we focused on those IPMNs without an invasive component (). Nine of fourteen patients were male (64.2%), and the mean age was 66 years old. Five of the IPMNs that we xenografted included an associated invasive cancer elsewhere in the lesion, and nine of the IPMNs were exclusively non-invasive. The non-invasive IPMN components were classified as high grade dysplasia in 7 cases (50%), moderate dysplasia in 5 (36%), and low-grade dysplasia in 2 (14%). The associated invasive component was a poorly-differentiated adenocarcinoma in 1 case (20%), a moderately-differentiated adenocarcinoma in 3 (60%), and a well-differentiated adenocarcinoma in 1 case (20%). The invasive adenocarcinomas were categorized as colloid type in one case and tubular type in 4 cases.
Clinicopathological Findings of Patients with IPMNs
The results of the IHC labeling for cytokeratin, MUC1, MUC2, p16/CDKN2A, TP53, and DPC4/SMAD4, on the primary neoplasms, are shown in . Most of the invasive IPMNs were the pancreatobiliary subtype, and produced associated tubular carcinomas (cases 1–4). Case 5 was the only intestinal subtype and formed a colloid carcinoma. In contrast, non-invasive IPMNs were either gastric or intestinal subtypes, except for case 8. Cytokeratin was positive and vimentin was negative for all cases. MUC expression patterns matched with the subtype, where MUC1 was positive for the pancreatobiliary type, and MUC2 was positive for the intestinal type (26
). All non-invasive IPMNs expressed p16/CDKN2A and DPC4/SMAD4, but were negative for TP53. The invasive IPMNs showed a loss of p16/CDKN2Aexpression in 3/5 cases, were TP53 positive in 1/5 cases. All 5 cases had intact DPC4/SMAD4.
Immunohistochemical profile of primary IPMNs
In vivo growth of human IPMNs in immunodeficient mice
We studied 14 total cases, of which only one was directly cultured (, case 11). Of the 13 IPMNs implanted in mice, one was implanted into a nude mouse, two were implanted into SCID mice, eight were implanted into triple immunodeficient NOG mice (, cases 2,3,4,5,6,7,9,14), and two were implanted into both SCID and NOG mice (, cases 8, 12). Of the 13 implanted IPMNs, 11 grew as tumors in mice, while 2 did not grow. We attempted to culture, after explanting, four of the 11 tumors that grew as xenografts (, cases 1, 5, 8, 9). The other seven mice died of infection or other causes before their tumors could be harvested.
Strategy and results of in vivo and in vitro propagation
In vitro growth of IPMNs after explanting from mice
Following expansion in the immunodeficient mice, we explanted four cases for in vitro growth (). Another 3 cases were directly cultured from the surgical pathology suite (2 of which were also grown in mice).
For case 1, the xenografted neoplasm formed a cyst. The fluid in the cyst was aspirated and cultured, and the resultant cell line was designated IPMN-1Asp, while the solid component of the same neoplasm was cultured and this cell line was designated IPMN-1T. Fibroblasts, which normally overgrow in such cultures, were removed using selective trypsinization (23
). Neoplastic cells were successfully purified to homogeneity in the first passage for both IPMN-1T and IPMN-1Asp.
We had several problems producing cell lines from the other six cases. In case 5, the neoplastic cells did not attach, although fibroblasts attached and grew. In cases 9 and 11, some neoplastic cells attached and grew initially, but the growth rate was too slow and fibroblasts overgrew the culture in 2 weeks. In cases 13 and 14, neoplastic cells grew poorly and gradually died in primary cultures.
In 5 cases we varied the basal media and substrate. Four types of basal medium were used, including MEM, DMEM, RPMI, and a 1:1 (volume:volume) mixture of MEM and RPMI supplemented as described in the methods. For basal medium, the results using MEM and DMEM were equivalent and consistently superior to RPMI. Five different substrates were used: uncoated tissue culture plastic, glass cover slips, tissue culture plastic coated with rat-tail collagen, matrigel, or polylysine. In all cases, rat-tail collagen coated flasks were much better than matrigel, polylysine, or uncoated flasks. Glass cover slips were the worst substrate. Fibroblast growth appeared to be stimulated on both matrigel and polylysine. Selective trypsinization (see methods) helped to prevent fibroblast overgrowth.
Histological comparison of primary tumors, xenografts, and IPMN-1 cell lines
For case 1, we compared the histological and immunohistochemical findings in the matched primary tumor, the first passage xenograft, and the cell line (, ). The primary tumor was a main duct type IPMN, with high-grade dysplasia. The subtype was focal oncocytic mixed with pancreatobiliary. Focally, the neoplastic cells formed small irregular nests in the extensive inflammatory stroma associated with the neoplasm, representing less than 1 mm microscopic invasion. The pathologic stage was T1N0MX. The first passage xenograft grew as an IPMN without invasion. Immunolabeling for cytokeratin was diffusely positive in all three samples (the primary tumor, the xenograft, and the cell line), while labeling for vimentin was consistently negative. The primary IPMN expressed MUC1, but did not express MUC2 (), and this pattern was maintained in the xenograft and the cell lines (). Immunolabeling for the p16/CDKN2A protein was generally lost in the primary tumor, in the xenograft, and in the cell lines, although there was some heterogeneous expression in the primary tumor (see supplemental figure 1
). The primary IPMN, the xenograft, and the cell lines did not stain with antibodies to the TP53 protein. Expression of the DPC4/SMAD4 protein was positive in the primary tumor, xenograft, and cell lines.
Figure 1 Histology and immunohistochemistry of matched primary tumor and corresponding xenograft for case 1. Hematoxylin and eosin (HE) and immunohistochemical labeling for cytokeratin, MUC1, MUC2, P16/cdkn2a, TP53, and DPC4/SMAD4. Negative region of P16/cdkn2a (more ...)
Phase microscopy and immunohistochemistry for the cell lines, IPMN-1T and IPMN-1Asp, stained for cytokeratin, MUC1, MUC2, p16/CDKN2A, TP53, and DPC4/SMAD4 (20X).
Case 8 was an IPMN with moderate dysplasia, and was composed of pancreatobiliary and gastric subtypes. It did not have an associated invasive component. For MUC1, the primary IPMN was focally positive, and the xenograft was consistently positive. For MUC2, both the primary tumor and the xenograft were negative. Immunolabeling for the TP53 protein was negative in the primary tumor and the xenograft. In both the primary tumor and xenograft P16 and DPC4/SMAD4 were intact (supplemental figure 2
). We were unsuccessful at establishing a cell line for the explanted cells from case 8.
Genetic Characterization of IPMN-1 cell lines
Both IPMN-1T and IPMN-1Asp were wildtype for Kras
. For TP53
, there was only a germline SNP (P72R) and no somatic mutations were detected. For p16/CDKN2A
, there was a homozygous deletion as demonstrated by MLPA (). This homozygous deletion is consistent with our inability to amplify by PCR any of the exons, despite multiple attempts, and with the loss of immnohistochemical labeling. For DPC4/SMAD4
, all exons sequenced as wildtype, and no abnormalities were detected by MLPA (data not shown). These findings are consistent with retention of expression shown by IHC labeling. The results of cell line IPMN-1T and IPMN-1Asp both matched a non-neoplastic sample from the patient using DNA fingerprinting (supplemental table 1
MLPA electropherograms of p16/CDKN2A. Negative wild type control (a). Positive control cell line with a homozygous deletion (b). IPMN-1T (c).
Tumorigenicity of the IPMN-1 cell lines
In anchorage-independent cloning assays, both IPMN-1Asp and IPMN-1T grew in soft agar and formed colonies at approximately equivalent rates. The frequency of colony formation was significantly less than that of the positive control Panc-1 cells (figure S3
), but higher than the negative control HPDE cells. We used invasion through matrigel to select an invasive subclone from the population of cells by plating them on top of matrigel coated filters, and growing the cultures until cells could be recovered from the bottom of the wells. Using this approach, invasive IPMN-1T cells were not detected until after 2 weeks of growth on the matrigel, whereas Panc-1 invaded by 6 hours. Invasive cells were expanded and the matrigel selection process was repeated.
We then tested the parental IPMN-1T, the second passage matrigel selected derivative (IPMN-1T-M2), and Panc1 in the standard matrigel assay, which measures cell number on the bottom of a matrigel coated filter at 6–72 hours (supplemental figure S4
). At 6 and 24 hour time points, no cells had invaded from either the IPMN-1T or IPMN-1T-M2 cells, in contrast to the Panc1 cells. After 48 hours, a few cells had invaded from both IPMN-1T and IPMN-1T-2M cultures.
In addition, to assess in vivo tumorigenicity, two second passage NOG mice were subcutaneously injected with 20 million cells bilaterally, and one tumor was detected approximately 12 weeks following injection that measured 7 × 6 mm (126 mm3, ). The tumor formed cysts and papillary structures as typically seen in IPMNs. Histology showed only non-invasive IPMN with high-grade dysplasia, with a pancreatobiliary subtype (). Half of this tumor was subcutaneously re-implanted into a third passage NOG mouse. After 14 weeks, the tumor had grown to 20×15mm (2250 mm3, ) and was explanted. The tumor formed a cystic mass, from which we aspirated approximately 4.5 ml of mucinous fluid. After aspirating the cyst fluid, the tumor was opened, and papillary nodules were revealed inside the cystic mass (). The histology () demonstrated an IPMN-like papillary structure with high-grade dysplasia, but without invasion.
Figure 4 IPMN-1 re-implantation. The tumor in NOG mouse approximately 12 weeks after injection with 20 million IPMN-1T cells (a). Histology of the reimplanted tumor (20X) showing a region without invasion (b, c). The third passage tumor in NOG mouse in 12 weeks (more ...)