Novel Pancreatic Cancer Cell Lines Derived from Genetically Engineered Mouse Models of Spontaneous Pancreatic Adenocarcinoma: Applications in Diagnosis and Therapy

Pancreatic cancer (PC) remains one of the most lethal human malignancies with poor prognosis. Despite all advances in preclinical research, there have not been significant translation of novel therapies into the clinics. The development of genetically engineered mouse (GEM) models that produce spontaneous pancreatic adenocarcinoma (PDAC) have increased our understanding of the pathogenesis of the disease. Although these PDAC mouse models are ideal for studying potential therapies and specific genetic mutations, there is a need for developing syngeneic cell lines from these models. In this study, we describe the successful establishment and characterization of three cell lines derived from two (PDAC) mouse models. The cell line UN-KC-6141 was derived from a pancreatic tumor of a KrasG12D;Pdx1-Cre (KC) mouse at 50 weeks of age, whereas UN-KPC-960 and UN-KPC-961 cell lines were derived from pancreatic tumors of KrasG12D;Trp53R172H;Pdx1-Cre (KPC) mice at 17 weeks of age. The cancer mutations of these parent mice carried over to the daughter cell lines (i.e. KrasG12D mutation was observed in all three cell lines while Trp53 mutation was observed only in KPC cell lines). The cell lines showed typical cobblestone epithelial morphology in culture, and unlike the previously established mouse PDAC cell line Panc02, expressed the ductal marker CK19. Furthermore, these cell lines expressed the epithelial-mesenchymal markers E-cadherin and N-cadherin, and also, Muc1 and Muc4 mucins. In addition, these cell lines were resistant to the chemotherapeutic drug Gemcitabine. Their implantation in vivo produced subcutaneous as well as tumors in the pancreas (orthotopic). The genetic mutations in these cell lines mimic the genetic compendium of human PDAC, which make them valuable models with a high potential of translational relevance for examining diagnostic markers and therapeutic drugs.


Introduction
Despite many advances in the understanding of molecular mechanisms involved in pancreatic cancer (PC) pathogenesis over the last four decades, the disease remains one of the top malignancies with worst prognosis [1]. These grim statistics are a constant reminder of the urgent need for elucidating yet undiscovered mechanisms of PC pathology that will contribute to improved diagnosis and treatment regimens. For this purpose, developing preclinical models is of vital importance, because they are critical for evaluating novel therapeutic strategies [2]. Xenograft tumors in athymic nude mice are useful preclinical models, but they cannot provide the role of immune mechanisms that may add to or interfere with the action of the therapeutic candidates. More recently, genetically engineered mice (GEM) models that produce spontaneous pancreatic adenocarcinomas (PDAC) have greatly advanced our understanding of PC pathogenesis and also, allowed the examination of novel therapeutic approaches [3][4][5][6]. In addition, syngeneic cell lines can be isolated from pancreatic tumors produced by GEM models and used for in vitro and in vivo screening assays. The analysis of functions and characteristics of specific genetic mutations and PC biomarkers present in these cell lines can shed light on the design of promising diagnostic and therapeutic strategies.
Mutations in KRAS, CDKN2A, TP53, and SMAD4/DPC4 genes are commonly observed in PDAC tumors from PC patients [7]. In consideration of these results, several mouse models that produce spontaneous PDAC, have been engineered in the last decade [3,4,6]. The present study focuses on mice carrying Kras and Trp53 mutations. The role of oncogenic Ras in PC was examined by directing endogenous expression of Kras G12D in the progenitor cells of the pancreas in Kras G12D ;Pdx1-Cre (KC) mice [3], whereas the role of the endogenous expression of Trp53 R172H and Kras G12D was examined in the pancreas of Kras G12D ;Trp53 R172H ;Pdx1-Cre (KPC) mice [4]. The results indicate that the spontaneous pancreatic tumors produced by these mouse models recapitulate the clinical, histopathological and genomic features of human PDAC.
Mouse PDAC cell lines with greater clinical relevance to PC are highly needed. The currently available Panc02 cell line has been used over the past three decades [8]. It was derived from PDAC tumors induced by implanting 3-methyl-cholanthrene (3-MCA)saturated threads of cotton in the pancreas of C57BL/6 mice. Despite its widespread use in evaluating various therapeutic strategies, Panc02 cells lack strong clinical significance for PC due to absence of mutational spectrum when compared to human disease. Consequently, success in translating therapies indicated by this model has been limited. In this manuscript, we describe the generation and characterization of three new PDAC cell lines derived from spontaneous mouse models of PC. One cell line was derived from a KC mouse at 50 weeks age, and two others were derived from KPC mice at 17 weeks of age. The successful establishment and in vitro and in vivo characterization of these cell lines are comprehensively described, including markers currently known for pancreatic tumors.
The collected tissues were washed 3 times with PBS-gentamicin and transferred into a petri dish containing the complete medium. The tumor was finely-minced with a sterile scalpel and transferred into a sterile centrifuge tube with the complete medium containing Collagenase P (Roche, Indianapolis, IN) (10 mg/ml).The mixture was incubated at 37uC for 30 min. The tubes were inverted every three min to ensure proper mixing. Following two washing cycles in complete medium, the pellet obtained after centrifugation was suspended in the complete medium. After letting the tube stand for 2 min, cell suspension without tissue debris was transferred into a new sterile 10 cm Petri dish.
After incubating overnight at 37uC in 5% CO 2 , the medium was replaced with fresh medium. Cells were trypsinized once confluent. To prevent the growth of fibroblasts, differential trypsinization was carried out, where trypsin was added to the cells and after 10 sec, the cells were washed with PBS and fresh medium was added. Additionally, medium was supplemented with cholera toxin (400 ng/ml) for the first 7 passages as it has been previously reported that it has an enhanced mitogenic effect on epithelial cells, but not on fibroblast cells [9]. After 10 passages, the cell lines were transferred to normal complete medium (i.e. DMEM medium supplemented with 10% FBS, 100 mg/ml penicillin, 100 mg/ml streptomycin) and maintained at 37uC and 5% CO 2 in a humidified incubator. Three cell lines were established successfully. The cell line derived from a Kras G12D ;Pdx1-Cre (KC) mouse was named UN-KC-6141 and the two cell lines derived from Kras G12D ;Trp53 R172H ;Pdx1-Cre (KPC) mice were named UN-KPC-960 and UN-KPC-961 (UN designates University of Nebraska Medical Center). In vitro characterization and tumorigenic studies were done after 35 passages in cell culture. The murine PDAC cell line Panc02 was included in the in vitro functional assays.

Growth Kinetics
For growth kinetics studies, each cell line was seeded in quadruplicate in a 96-well plate (1,000 cells/well) in complete medium. After overnight incubation, the medium was replaced with medium supplemented with 1% FBS and Penicillin/ Streptomycin (100 mg/ml). Every day, 10 ml of the Cell Proliferation Reagent WST-1 (Roche, Indianapolis, IN) was added to each well and after 3 h of incubation, absorbance values were measured at 450 nm. Absorbance values were subtracted from values recorded at the reference wavelength (600 nm), as indicated by the manufacturer. The procedure was repeated for 7 days. The doubling time (T D ) for each cell line was calculated during the exponential growth phase using the formula T D = 0.693t/ln(N t / N 0 ), where t = time difference in h, N t = absorbance value at time t, and N 0 = absorbance value at initial time [10].

Real Time PCR
RNA was isolated and purified from UN-KC-6141, UN-KPC-960, UN-KPC-961, Panc02, and NIH3T3 (mouse fibroblasts) cells using the RNeasy Minikit (Qiagen, Germantown, MD). cDNA was synthesized from total RNA using oligo(dT)18 primer and SuperScript II reverse transcriptase (Life technologies TM , Carlsbad, CA) as described by us in a previous publication [11]. Realtime PCR was carried out in the LightCycler 480 (Roche Diagnostics, Indianapolis, IN). The amplification was done in a two-step process (95uC for 5 min followed by 45 cycles of 95uC for 10 sec, 60uC for 10 sec, and 72uC for 10 sec) using the LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics, Indianapolis, IN) and cDNA from each sample. GAPDH was used as the internal control gene to which all genes were normalized. The fold-change in gene expression of Amylase and CK19 mRNA in cancer cell lines were compared relative to mRNA levels extracted from normal mouse pancreas using the 2 2DDCt method, whereas the foldchange of Muc1 and Muc4 were compared relative to mRNA extracted from the mouse fibroblast cell line NIH3T3. The sequence of the primers used in this study are listed in Table 1.

Antibodies
The anti-CK19 hybridoma (TROMA-III) developed by Dr. Rolf Kemler was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained at The University of Iowa (Iowa City, IA). Antibody against murine E-cadherin was obtained from Cell Signaling (Danvers, MA). N-cadherin antibody was a kind gift from Dr. Keith R. Johnson from UNMC (Omaha, NE). b-actin and Amylase antibodies were purchased from Sigma Aldrich (St. Louis, MO). For confocal studies the secondary antibodies conjugated to Alexa FluorH (568, 488) were obtained from Life Technologies TM (Carlsbad, CA). The secondary antibodies conjugated to horseradish peroxidase used for western blot analysis were obtained from GE Healthcare Life Sciences (Uppsala, Sweden). Anti-mouse Muc1 antibody (mouse monoclonal antibody recognizing the cytoplasmic tail of Muc1) was purchased from AbcamH (Cambridge, MA, USA). The anti-Muc4 (4A-rabbit polyclonal) antibody was designed in this lab and developed by GenScript (Piscataway, NJ, USA) as has been described previously [12].

Confocal Microscopy
Protein expression was analyzed by confocal microscopy. 2610 5 cells suspended in complete medium were seeded on glass cover slips placed in a 12-well plate. The next day, cells were fixed in ice cold methanol. After washing in PBST and blocking with 10% goat serum (Jackson Immunoresearch Labs, West Grove, PA), the cells were incubated with antibodies for Amylase (1:500), CK19 (1:1,000), E-cadherin (1:50), N-cadherin (1:20), and Muc1 (1:100) overnight. They were washed four times with PBST, 5 min per wash. The cells were incubated with the respective secondary antibodies (mouse/rabbit) conjugated to Alexa Fluor (568, 488) (Life Technologies TM , Carlsbad, CA) and after repeating the washing steps, the glass coverslips were mounted on glass slides with vectashield mounting medium (Vector Laboratories, Burlingame, CA). The cells were imaged on a laser confocal microscope LSM 510 (Carl Zeiss GmbH, Thornwood, NY) in the respective wavelengths at a magnification of 663.

Western Blot Analysis
For western blot analysis, cells were seeded on each well of a 6well plate in complete medium. At ,80% confluency, cells were lysed with radioimmunoprecipitation assay buffer (RIPA) containing protease and phosphatase inhibitors. The lysates were subjected to several freeze-thaw cycles to ensure complete lysis. The concentration of lysates was determined with the micro-bicinchoninic acid protein estimation kit (Bio-Rad, Hercules, CA). The protein concentrations were adjusted and solutions were prepared under reducing conditions (i.e. b-mercaptoethanol). 2% SDS-agarose gels were used for analysis of Muc4 expression. 40 mg of protein lysates were loaded and the gel was run for 4 h at 100 V. E-cadherin and N-cadherin expression levels were analyzed by SDS-PAGE. 40 mg of protein lysates were loaded and separation was done at 80 mA for 1 h. Resolved proteins were transferred onto polyvinylidene difluoride membranes, blocked in 5% milk in PBS, and incubated with the primary antibodies overnight. After washing with PBST, the corresponding secondary antibodies were added and after repeating the washing steps, the proteins were detected by luminol (Thermo Scientific, Middletown, VA) after exposure to X-ray films.

Cytotoxic Assay
The cytotoxic effect of the chemotherapeutic drug Gemcitabine on KC and KPC cell lines was compared to the cytotoxic effect on

Tumorigenicity studies
The tumorigenicity of the cell lines was determined after orthotopic implantation of the mouse PC cell lines (UN-KC-6141/ UN-KPC-960/UN-KPC-961) into the head of the pancreas after 35 passages. Based on the mice background from where the cell lines were generated, UN-KC-6141 cells (1610 6 ) were injected in C57BL/6 mice, whereas UN-KPC-960 and UN-KPC-961 cells (1610 6 ) were injected in mixed background (i.e. B6.129) mice (N = 7). Mouse PC cells suspended in 50 ml sterile PBS were injected orthotopically using the same procedure described by us [13,14]. Subcutaneous tumor growth was evaluated with the UN-KPC-961 cell line only. 5610 6 cells were injected (N = 12) in mixed B6.129 background mice on the lateral chest. Tumor growth was monitored by palpation/Vernier caliper measurements in case of subcutaneous tumors. Throughout the experiment, animals were provided with food and water ad libitum and subjected to a 12-h dark/light cycle. Animal studies were performed in accordance with the U.S. Public Health Service "Guidelines for the Care and Use of Laboratory Animals" under an approved protocol by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (IACUC). After euthanization, pancreatic tumors were dissected out, weighed and fixed in 10% formalin (Fisher Scientific, Fair Lawn, NJ) for H&E staining. Gross metastatic lesions were examined in distant organs and processed for histological analysis.

Hematoxylin and eosin staining (H&E)
After fixing tissues in 10% formalin for at least 48 h, the tissues were embedded in paraffin and serial tissue sections (4 mm thick) were cut. The sections were deparaffinized using EZ-DeWaxTM (Bio genex, San Roman CA, USA) and rehydrated progressively. Afterwards, the sections were stained with hematoxylin and eosin (H&E) stains and examined by a certified pathologist.

Statistics
Significant differences in the experimental values were determined by calculating p-values using the JMPH Statistical Discovery Software (Cary, NC). A Student's t-test was used to calculate the corresponding p-value (p-values , 0.05 were considered statistically significant).

In vitro establishment of KC and KPC cell lines
Mouse PC cell lines were successfully generated from the spontaneous pancreatic tumors produced by the Kras G12D ;Pdx1-Cre (KC) and the Kras G12D ;Trp53 R172H ;Pdx1-Cre (KPC) mice at 50 weeks and 17 weeks of age, respectively. One cell line derived from the KC mice was named UN-KC-6141, and two other cell lines derived from the KPC mice were named UN-KPC-960 and UN-KPC-961. All three cell lines grew for over 50 passages in normal media with no sign of senescence. Monolayer cultures of all three cell lines showed typical cobblestone morphology with closed contact island formation (Fig 1A). To verify that these cell lines maintained the genetic mutations of their respective parental mice (KC and KPC), the mutational status of the Kras (Fig. 1B) and Trp53 (Fig. 1C) loci was examined. As expected, all three cell lines carried the Kras G12D point mutation, whereas UN-KPC-960 and UN-KPC-961 carried the Trp53 R172H activating mutation.
The growth kinetics of these three mouse PC cell lines were compared with Panc02 cells (Fig 2A).

KC and KPC-derived cell lines have ductal-like characteristics
PDACs are thought to arise from the epithelial cells of the pancreatic duct [7]. The exocrine pancreas contains both acinar and ductal cells. Pancreatic acinar cells are characterized by amylase expression and lack of expression of either cytokeratin 19 (CK19) or mucins, and the ductal cells are characterized by expression of CK19 and mucins but no expression of amylase [15,16]. Indeed, previous studies have reported pancreatic tumors from KC and KPC mice expressed CK19 and frequently, mucin, indicating their ductal heritage [3,4]. To validate whether our KC and KPC derived-cell lines had these ductal characteristics, we examined amylase and CK19 expression by real time PCR and confocal microscopy. Panc02 cells were included in these analyses for comparison, and normal pancreatic tissue was used for estimating relative gene expression. Real-time PCR experiments showed high expression of the CK19 ductal marker in our three new PDAC cell lines (p , 0.005) compared to Panc02 (Fig. 3A). To our surprise, Panc02 cells did not show any CK19 expression, even though it has been referenced as a murine ductal PDAC cell line for nearly three decades [8]. None of the four cell lines showed amylase expression, whereas normal pancreas expressed this acinar marker. These results were further confirmed by confocal microscopy analysis (Fig. 3B).

KC and KPC cell lines have epithelial-mesenchymal characteristics
The epithelial-mesenchymal transition induces loss of cell adhesion, which corresponds to E-cadherin downregulation and increased expression of N-cadherin, leading to the initiation of PDAC metastasis [17]. We examined E-cadherin and N-cadherin expression by confocal microscopy and western blot analyses in the three newly-established KC and KPC cell lines. E-cadherin expression was observed in UN-KC-6141, UN-KPC-960, and UN-KPC-961 cells, indicative of their epithelial nature (Fig. 4A-B). In contrast, Panc02 cells showed minimal E-cadherin expression. The pattern of N-cadherin expression was similar, being more prominent in the KC and KPC cell lines, compared to the expression in Panc02 cells (Fig. 4C-D).

KC and KPC cell lines express high levels of Muc1 and Muc4
Different types of mucins are expressed in the pancreas during PC progression [12,18]. Furthermore, PC precursor lesions in genetically engineered mouse PDAC models such as the KC and KPC mice, are known to produce mucins [3,4]. Recently, we also have demonstrated that in the pancreas of KC mice, expression of Muc1, Muc4, and Muc5AC progressively increased in correlation with PDAC development [12]. We examined whether the expression of these mucins can be corroborated in the KC and KPC-derived cell lines. To this end, real-time PCR, western blot, and confocal microscopy analyses were conducted. The results indicated that relative transcript levels of the transmembrane mucins Muc1 (p , 0.001) and Muc4 (p , 0.05) were significantly  (Fig. 5A) compared to control cells (NIH3T3 fibroblasts). In Panc02 cells, the relative transcript levels of Muc1 and Muc4 were elevated, but they were not statistically significant. Nevertheless, Panc02 cells and the two cell lines derived from KPC mice showed higher levels of Muc4 protein than the KC-derived cell line (Fig. 5B). On the other hand, Muc1 protein in Panc02 was lower than in the KC and KPC-derived cells (Fig. 5C). To our surprise, Muc5AC was not detectable in any of the cell lines, either at transcript or at protein levels (data not shown), and this may be a consequence of evolutionary changes associated with cell culture.

KC and KPC cell lines are resistant to Gemcitabine
Despite recent reports that PC patients show greater survival when treated with FOLFIRINOX (a combination of oxaliplatin, irinotecan, fluorouracil and leucovorin) instead of Gemcitabine  [19], the latter is still the most used drug in PC chemotherapy [20]. Therefore, using the MTT assay, we examined Gemcitabine cytotoxicity on UN-KC-6141, UN-KPC-960, UN-KPC-961 and Panc02 cells (Fig. 6A). Panc02 cells were the most Gemcitabineresistant with a half maximal inhibitory concentration (IC-50) of 15 mM after 48 h of treatment (Fig. 6B). The IC-50 values of the KC and KPC cell lines ranged from 0.5 mM to 5 mM. Although Panc02 cells were more resistant to Gemcitabine, these IC-50 values are in the comparable range of human PC cells [21,22]. Interestingly, at higher Gemcitabine concentrations (30-100 mM) (Fig. 6A), the UN-KPC-961 cells showed greater Gemcitabine resistance than the Panc02 cells.   formed tumors after orthotopic implantation (Fig. 7A), but those appeared at slower rates and the tumors took over two months to grow. The comparatively slower tumor growth kinetics of the KPC cells may have been caused by genetic differences between KC and KPC cells, as well as the different backgrounds of their respective host mice. Interestingly, the UN-KPC-961 cells appeared to be more tumorigenic (tumor incidence 86%) than the UN-KPC-960 cells (tumor incidence 43%). These results are in agreement with their growth kinetics (i.e. UN-KPC-961 showed faster growth than UN-KPC-960). In addition, UN-KPC-961derived tumors appeared to be more aggressive, showing metastasis to spleen, stomach, liver, and diaphragm, whereas the UN-KPC-960-derived tumors did not show any metastatic lesions. When UN-KPC-961 PC cells were injected subcutaneously into the mixed B6.129 background mice, all mice developed tumors in three weeks, but no metastases were observed. Figure 7A shows a comparison of tumors derived from these experiments. Although the average weights of the orthotopic tumors derived by the UN-KPC-960 cells were smaller, tumor weight differences across all groups were not statistically significant. Further, we also performed subcutaneous injection using UN-KC-6141 and UN-KPC-960 PC cells into two C57BL/6 and B6.129 background mice and all the mice developed tumors after 4 weeks (data not included). For PDAC, histological grading is assigned by the extent of glandular differentiation [23]. If over 95% of a tumor is composed of glands then it is classified as well differentiated, if that extent is 50-95%, a tumor is considered moderately differentiated, and if less than 50%, a tumor is described as poorly differentiated. Histologic analyses classified the KC and KPC-derived tumors as moderately to poorly differentiated tumors, because they did not show duct formation (Fig. 7B). In fact, most of these tumors presented characteristics of poorly differentiated (grade III) adenocarcinoma. These results are different from previous studies reporting that most spontaneous pancreatic tumors from KC and KPC mice show moderately well-differentiated to well-differentiated morphology [3,4].

Discussion
Genetically engineered mouse models are presently a promising approach for understanding the pathogenesis and progression of PDAC and for evaluating novel therapeutic agents (natural/ synthetic) that should translate to high clinical success [24]. Unfortunately, the generation and maintenance of these advanced models require a high cost and time investment. PDAC cell lines from these model mice should have a major role in facilitating screening and prioritization of the variables (e.g. study gene roles or novel therapies), before the ideas are evaluated in the more expensive mouse models.
To our knowledge, this is the first paper reporting the successful establishment and characterization of PDAC cell lines derived from Kras G12D ;Pdx1-Cre (KC) and Kras G12D ;Trp53 R172H ;Pdx1-Cre (KPC) mouse models. The three cell lines derived in these studies, named UN-KC-6141, UN-KPC-960, and UN-KPC-961, have been maintained in cell culture for over 50 passages without any sign of senescence. These cell lines show typical epithelial cobblestone morphology, and expressed high levels of epithelial and mesenchymal markers such as CK19, E-cadherin, Ncadherin, Muc1, and Muc4 ( Table 2). Although Panc02 has been classified as a PDAC cell line on the basis of histological analysis since its establishment three decades ago [8], we noted with interest that it does not express the ductal marker CK19, which raises questions about its phenotype ( Table 2). Furthermore, unlike the KC and KPC cell lines, Panc02 cells did not express E-cadherin and N-cadherin. As differential levels of these cadherins have a major role in metastasis [17], these cell lines should be useful for evaluating various therapeutic agents or study the fuction of various genes by manipulation.
The most commonly occurring mutations in PDAC include those in the KRAS, CDKN2A, TP53, and SMAD4/DPC4 genes [7]. Mutations in the KRAS gene are present in 90% of PDAC [25], TP53 mutations occur in ,75% of PDAC [26], and 55% of pancreatic tumors have SMAD4/DPC4 deletions or mutations [27]. The KC and KPC-derived cell lines described here did not suffer from evolutionary changes in their cancer genes. Sequencing of these cell lines for point mutations in KRAS and P53 genes revealed the presence of Kras point mutations (Gly-Asp) in all the KC and KPC-derived cell lines, and the Trp53 mutation (R172H) was only present only in the KPC cell lines. Two recent publications have reported the well-established Panc02 cell line has a Smad4 mutation, but both Kras and Trp53 mutations are absent [28,29]. Considering that human PDAC show higher frequency of KRAS and TP53 mutations and ductal characteristics, the newly derived murine PDAC cell lines UN-KC-6141, UN-KPC-960, and UN-KPC-961 should be very valuable preclinical studies and have excellent translational significance.
Gemcitabine is the most used chemotherapeutic treatment for PC patients, but tumors may acquire resistance, as evidenced by the poor survival statistics [30]. Some studies have shown that epithelial to mesenchymal transition contributes to drug resistance in PC cells [31,32]. In one of these studies it was documented that E-cadherin levels in cancer cells have an inverse correlation with drug resistance [32]. This may explain why Panc02 was more resistant to the effects of Gemcitabine (IC-50 = 15 mM) when compared to the spontaneous PDAC cell lines (IC-50s = 0.5-5 mM). The expression levels of E-cadherin in UN-KC-6141, UN-KPC-960, and UN-KPC-961 cells correlated with decreased drug resistance. It is important to note that even though the KC and KPC-derived PDAC cell lines were not as resistant to Gemcitabine as the Panc02 cells, these cytotoxicity levels are in the range of those found in human PDAC cell lines [21,22].
In conclusion, the UN-KC-6141, UN-KPC-960, and UN-KPC-961 cells described here represent a valuable tool for yielding a better understanding of PC pathogenesis and to study novel treatments for this lethal disease. The genotypic and phenotypic characteristics of these novel cell lines mimic human PDAC, which make them valuable models with significant translational relevance.