Development of syngeneic murine cell lines for use in immunocompetent orthotopic lung cancer models

Background Immunocompetent animal models are required to study tumor-host interactions, immunotherapy, and immunotherapeutic combinations, however the currently available immunocompetent lung cancer models have substantial limitations. While orthotopic models potentially help fill this gap, the utility of these models has been limited by the very small number of murine lung cancer cell lines capable of forming orthotopic tumors in immunocompetent C57BL/6 hosts. Methods Primary lung tumors with specific genetic alterations were created in C57BL/6 background mice. These tumors were then passaged through other animals to increase tumorigenicity and select for the ability to grow in a non-self animal. Once tumors demonstrated growth in a non-self host, cell lines were established. Successful cell lines were evaluated for the ability to produce orthotopic lung tumors in immunocompetent hosts. Results We produced six murine lung cancer lines capable of orthotopic lung tumor formation in immunocompetent C57BL/6 animals. These lines demonstrate the expected genetic alterations based on their primary tumor genetics. Conclusions These novel cell lines will be useful for evaluating tumor-host interactions, the impact of specific oncogenic alterations on the tumor microenvironment, and immunotherapeutic approaches. This method of generating murine lines capable of orthotopic growth can likely be applied to other tumors and will broaden the applicability of pre-clinical testing of immunotherapeutic treatment regimens.


Background
Although immunotherapy is the biggest treatment advance in metastatic lung cancer in over 30 years most patients do not respond to this approach and a better basic understanding of tumor-immune interactions is required for immunotherapy to reach its full potential [1]. Unfortunately, the immunocompetent animal models required for these studies are extremely limited. While genetically engineered mouse models (GEMMs) produce tumors in an immunocompetent background, many GEMMs generate multifocal tumors of low malignant potential that may not accurately recapitulate the complex tumor-host interactions present during disease progression [2]. In addition, the low mutational burden of GEMM tumors may limit their utility for studying immunotherapy where therapeutic response is partially dependent on

Open Access
Cancer Cell International *Correspondence: Stephen.Malkoski@cuanschutz.edu 1 Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver Anschutz Medical Campus, 12700 E. 19th Avenue, RC2, Room #9112, Mail stop C272, Aurora, CO 80045, USA Full list of author information is available at the end of the article tumor neoantigens [3,4]. That the viral vectors commonly used to initiate tumor formation also transduce resident immune cells further complicates the use of these models [5,6]. Finally, generating tumors and monitoring therapeutic responses in GEMMs is complicated and costly.
Orthotopic systems where tumor cells are directly injected into the lungs of recipient mice can also be used to model tumor-host interactions. While this better models metastatic disease and allows for significantly shorter studies then GEMMs [7], this approach has been limited by the small number of transplantable murine lung cancer cell lines. To the best of our knowledge, there are only two commercially available C57BL/6 derived murine lung tumor lines capable of forming orthotopic lung tumors in immunocompetent hosts. The Lewis Lung Carcinoma (LLC) line was subcloned from a spontaneous lung tumor in 1951 [8,9] while CMT167 was sub cloned for metastatic potential from the CMT64 line derived from a spontaneous lung tumor in 1976 [10,11]. More recently, GEMM-derived lines developed in a mixed genetic background have been described [12][13][14], however the broad utility of these lines is unclear as these lines may have limited tumorigenicity in C57BL/6 mice. An exception is a Kras G12D .p53 −/− line derived in a C57BL/6 background that forms lung tumors in C57BL/6 mice after tail vein injection [15]. The development of lines capable of orthotopic growth specifically in a C57BL/6 host is critical as many genetic tools for manipulating the murine immune system in vivo exist in this background.
In addition, all the above mentioned cell lines harbor activating Kras mutations [12][13][14][15][16] which may limit generalizability to other oncogenic drivers. Although the mechanistic relationships between oncogenic drivers and immunotherapeutic response remains unclear, human tumors with targetable oncogenic drivers appear poorly responsive to programmed death ligand 1 (PD-L1) blockade [17]. Moreover, the best characterized murine lung cancer cell lines (CMT and LLC) have disparate responses to programmed death ligand-1 (PD-L1) blockade [16], suggesting it will be difficult to discern the relationship between oncogenic driver and immunotherapeutic response without substantial additional tools. Herein, we describe a process for developing murine lung cancer cell lines with a variety of genetic alterations that are capable of forming orthotopic lung tumors in C57BL/6 hosts. This approach will facilitate assessment of tumor-host interactions in the context of different genetic drivers. These lines will be useful for testing combinations of chemotherapy, immunotherapy, and radiation therapy in preclinical models.

Primary tumor formation
Adenovirus with Cre recombinase under the control of the cytomegalovirus (CMV) promoter (Ad5-CMV-Cre) or the surfactant protein C (SPC) promoter (Ad5-SPC-Cre) was purchased from the University of Iowa Viral Vector Core (Iowa City, IA). Adenovirus capable of mediating the echinoderm microtubule-associated protein-like 4 (EML4) anaplastic lymphoma kinase (ALK) gene fusion (Ad-EA) [26] was purchased from Viraquest (North Liberty, IA) with the permission of Dr. Andrea Ventura (Memorial Sloan Kettering). Tumor formation was initiated by injecting 2 µl of virus directly into the left lung or by tracheal instillation as previously described [27,28] and as detailed in the Results. Cre recombinase viruses were used in animals harboring alleles for conditional oncogene knock-in and/or conditional tumor suppressor deletion while Ad-EA was used in C57BL/6 wild type mice. Animals harboring primary tumors were euthanized 11-36 weeks after tumor initiation.

Tumor passaging
Primary tumors were dissected from surrounding lung tissue then minced with razor blades. An aliquot of the minced homogenate was suspended in Hanks' Balanced Salt Solution (HBSS, 14170-112, Gibco, Grand Island, NY) supplemented with 1.3 mg/ml Matrigel (#354234, Corning, Oneonta, NY); 40 µl of this suspension was injected into the left lung of a recipient animal as previously described [7] while 400 µl was injected into the right flank of the same recipient. Animals harboring transplanted tumors were monitored until flank tumor size exceeded 1 cm or until animals showed signs suggestive of internal tumor burden (weight loss, hunched posture) or for up to 9 months. At this point, recipient animals were euthanized and tumors collected for passaging as described above and culture as described below. At each passage, lung tumors > 5 mm were passaged separately (i.e., into separate recipient animals) from flank tumors while lung tumors < 5 mm were combined with flank tumors from the same animal and passaged together (i.e., into the same recipient animal). To reduce the probability of rejection, sex matched recipients were used and all tumor recipients were genetically > 90% C57BL/6 by SNP analysis.

Assay for orthotopic tumor formation
Once lines were established in vitro, they were mycoplasma tested, treated if positive, and used between passages 5-10 for orthotopic experiments. Cell suspensions in 50% HBSS/50% Matrigel were created then 500,000 cells in 400 µl were injected into the right flank and 250,000 cells in 40 µl were injected into the left lung as previously described [7]. Recipient mice were monitored until flank tumors exceeded 1 cm or until animals showed signs of internal tumor burden (weight loss, hunched posture) or for up to 45 days. If flank tumors developed too quickly to reliably evaluate lung tumor formation, flank and lung tumor formation was assessed in separate animals. At least four animals (two male and two female) were used to determine the tumorigenicity of each cell line. Lines were deemed successful if they formed tumors in at least 75% of recipient animals.
To assess GFP expression, cultured cells were heat fixed (95 °C for 5 min) to glass slides, counterstained with DAPI and examined at 510 nm. Western blotting for pAKT and pERK after crizotinib (Selleck, Houston, TX) treatment was performed as previously described [34] using the following antibodies: pAKT S437 (Cell Signaling #4058), total AKT (Cell Signaling #2920), pERK1/2 T202/Y204 (Cell Signaling clone D13.14.4E), total  TAG GTG TTG GGA TAG CTG  TCC GAA TTC AGT GAC TAC AGA TGT ACA GAG   PTEN  ACT CAA GGC AGG GAT GAG C  AAT CTA GGG CCT CTT GTG CC  GCT TGA TAT CGA ATT CCT GCAGC   SMAD4  TCC CAC ATT CCT CTT AGT TTTGA  CCA GCT TCT CTG TCC AGG TAGTA   PIK3CA  CAC AGC TCG CGG TTG AGG  TGC TCG ACG TTG TCA CTG AA  CGG GTG TAC TCC TCA TAT AACA   TGFβR2  AGG GAT GAA TGG GCT TGC TT  CTC ACC TCA GAG CCT GAT TA   TAK1  GCA ACT TCG ACA ACT TGC CTT CCT GTG  GCA CTT GAA TTA GCG GCC GCA AGC TTA TAA CT   EML-ALK  GAG CCT TGT TGA TAC ATC GTTC  TAG GAG GCA GTT TGG GCT AC CAA GGC AGT GAG AAC CTG AA ERK1/2 (Cell Signaling clone L34F12). In vitro cell viability assay was performed as previously described [35]. Briefly, cells were plated into 96-well plates at 1000 cells/ well 24 h prior to drug treatment then treated with serial dilutions of the ALK inhibitor TAE-684 (Selleck) for 72 h and viability determined by MTS assay (CellTiter96 AQueous Kit, Promega). Percent inhibition and IC50 were calculated using GraphPad.

General approach to development of syngeneic murine lines with orthotopic growth potential
We generated mice for primary tumor formation using combinations of conditionally activated tumor-initiating oncogenes (Kras LSL-G12D or R26Stop FL P110*) and conditionally deleted tumor suppressor alleles (Smad4 flox , Tgfbr2 flox , Map3k7flox, PTEN flox , p53 flox ; an example is shown in Fig. 1a). Specific oncogene/tumor suppressor combinations were selected based on prior work [36][37][38] and ongoing projects. Tumors were initiated by injecting adenovirus that expresses Cre recombinase directly into the left lung [27,28]. Upon Cre recombinase exposure, oncogenes are activated via excision of an upstream loxPstop-loxP sequence while tumor suppressors are deleted via LoxP sites surrounding exons. Some animals also harbored a tracking allele (ROSA mTmG ) in which cells switch from expressing mTomato to mGFP after recombination; an example of a primary tumor is shown in Fig. 1b.
To enhance the development of tumor lines that could grow in a non-self host, primary tumors were passaged through the flanks and lungs of recipient animals. At each passage after P1, we attempted to establish cell lines from passaged tumors. Once cell lines were established, we tested their ability to form orthotopic tumors. If a cell line was capable of forming orthotopic tumors, we assessed the line for the expected genetic and molecular changes as described in methods and shown in subsequent figures. Workflow is shown in Fig. 1c. Generation of these lines was time intensive, taking 300-500 days from the time that primary tumors were initiated through the time that orthotopic tumor formation was established ( Table 2); this excludes time required for breeding and genotyping animals prior to tumor initiation and time for validating lines after orthotopic tumor formation ability was established. This process is also relatively inefficient with only 5% (6/113) primary tumors ultimately leading to lines capable of orthotopic tumor formation (Fig. 1d). Interestingly, the majority of failures (77%; 87/113) occurred at P1; if a successful P1 tumor was established, 6/26 (23%) tumors ultimately led to a cell line capable of orthotopic tumor formation. G12D .Smad4 +/− cell line A 6 week old Kras LSL-G12D/+ .Smad4 fl/+ male mouse was injected with 2 µl of 10 10 PFU/ml Ad5-CMV-Cre into the left lung. When this animal was euthanized 26 week later, a 12 mm primary tumor was passaged into a male recipient animal. After 3 passages through recipient animals (two of which included small lung tumors), cell line X577 was established that was capable of forming orthotopic tumors in immunocompetent C57BL/6 animals (Fig. 2a). As expected, X577 cells demonstrate genetic recombination at the Kras and Smad4 loci (Fig. 2b). By Western blot, X577 cells express KRAS G12D but not SMAD4 (Fig. 2c).

Development and validation of a Kras G12D .Tgfbr2 −/− cell line
A 6 wk old Kras LSL-G12D/+ .Tgfbr2 fl/fl male mouse was injected with 2 µl of 10 10 PFU/ml Ad5-SPC-Cre into the left lung. When this animal was euthanized 29 week later, a 9 mm primary tumor was passaged into a male recipient. After two passages through recipient animals (one of which included a lung tumor), cell line X911 was established that was capable of forming orthotopic tumors in C57BL/6 animals (Fig. 3a). As expected, X911 cells exhibit genetic recombination at the Kras and Tgfbr2 loci (Fig. 3b) and express KRAS G12D but not TGFBR2 (Fig. 3c).    recipient animals, cell line E889 was established that was capable of forming orthotopic tumors in C57BL/6 animals (Fig. 4a). This line demonstrates genetic recombination at the Kras and Map3k7 loci (Fig. 4b) and expresses KRAS G12D but not MAP3K7 (Fig. 4c) by Western blot. Because this line was derived from an animal harboring the ROSA mTmG tracking allele [25], it also expresses GFP (Fig. 4d).

Development and validation of a Kras G12D .PTEN +/− .p53 +/− . GFP + cell line
A 7 week old Kras LSL-G12D/+ .PTEN fl/+ .p53 fl/+ .ROSA mTmG male mouse was injected with 2 µl of 10 10 PFU/ml Ad5-SPC-Cre into the left lung. When this animal was euthanized 19 week later, multiple primary tumors between 1 and 3 mm were passaged together into a recipient animal. After 3 passages two of which were through the lung, cell line X381 was established that was capable of forming orthotopic tumors in C57BL/6 animals (Fig. 5a). This line demonstrates genetic recombination at the Kras, Pten, and Tp53 loci (Fig. 5b). As expected, X381 cells express KRAS G12D but have reduced expression of PTEN and TP53 (Fig. 5c). Because this line was derived from an animal harboring the ROSA mTmG tracking allele, it also expresses mGFP (Fig. 5d).  [16], the 265 bp band in the Kras PCR from CMT and LLC cells represents the wild type (non-engineered) Kras allele. c Western blot showing KRAS G12D expression and SMAD4 loss in X577 cells. The KRAS G12D -specific antibody detects the KRAS G12V mutation in CMT cells but not the KRAS G12C mutation in LLC cells. The complete absence of SMAD4 expression suggests that the wild type Smad4 allele has undergone mutation, loss of heterozygosity, or transcriptional silencing then passaged into a female recipient animal. After one passage through the flank, cell line Y856 was established; this line was capable of forming orthotopic tumors in C57BL/6 animals (Fig. 6a). Y856 demonstrates genetic recombination of the Pik3ca and Tp53 alleles (Fig. 6b) and reduced TP53 expression (Fig. 6c). Consistent with constitutive PIK3CA activation, Y856 cells demonstrate increase pAKT expression without increased total AKT (Fig. 6c).

Development and validation of an EML4-ALK mutant cell line
An 8 wk old C57BL/6 female mouse was treated with 30 µl of 10 6 PFU/ml Ad-EA by tracheal instillation as previously described [27,28]. The Ad-EA vector has an Ad5 backbone and harbors Cas9 and guide RNAs that lead to the EML4-ALK gene fusion [26]. When this animal was euthanized 14 week later a group of multifocal tumors 3-5 mm in size were combined and passaged into a recipient animal. For this line, tumors formed in both lung and flank; these tumors were combined and then passaged together into both lung and flank sites of recipient animals. Subsequently, a cell line (Y143) was established that was capable of forming orthotopic tumors in C57BL/6 animals (Fig. 7a). The EML4-ALK genetic rearrangement was validated using PCR as previously described [26] (Fig. 7b). In Y143 cells treatment with the EML4-ALK inhibitor crizotinib inhibits phosphorylation of AKT and ERK is inhibited in a dose dependent manner (Fig. 7c) and treatment with the ALK kinase inhibitor, TAE684, inhibits growth of Y143 cells (Fig. 7d).

Rationale for developing syngeneic murine lung cancer cell lines
Better immunocompetent murine lung cancer models are required to study tumor-immune interactions and optimize immunotherapeutic approaches. GEMMs are limited by the production of multifocal tumors of relatively low malignant potential with limited mutational burden [2][3][4] while orthotopic models are limited by the small number of transplantable murine lung cancer cell lines and limited diversity of driver mutations. Our goal was to develop a systematic approach for producing murine lung cancer cell lines with different genetic alterations that were capable of orthotopic tumor formation in C57BL/6 background recipients. This is of particular relevance as most immune system genetic models exist in a C57BL/6 background and changing the genetic background is labor intensive and expensive. Table 2 summarizes the developmental details of our six novel murine lung cancer lines and illustrates that this process is time intensive, requiring 10-18 months from tumor initiation plus additional time to generate animals for primary tumor formation and validate cell line genetics.
Because of the length required to establish successful cell lines, these lines assuredly acquired other genetic alterations that contribute to cell survival, immune evasion, or other characteristics typical of cancers. Transcriptome characterization of lines during development could provide interesting insight into the common pathways required for both in vivo tumor formation and in vitro propagation.

Developing syngeneic murine lung cancer cell lines: maximizing primary tumor malignancy
Although Kras LSL-G12D/+ mice treated with tracheal Ad5-CMV-Cre expire 2-4 months of tumor initiation with lungs that are several times normal in size, most tumors are small adenomas or well differentiated adenocarcinomas [18]. This may explain our limited success in generating tumor lines from Kras LSL-G12D/+ animals (not shown). To address this issue, we initiated primary tumor formation via direct injection of virus into the left lung [27]. While tumor production takes significantly longer (4-8 months depending on genotype and virus), this approach allows the development of significantly larger and presumably more malignant tumors. Perhaps not surprisingly, half of our tumor lines originated from primary tumors that were   3, 4, 5, 6, 7a). This is consistent the observation that most GEMMs produce predominantly adenocarcinoma spectrum tumors.

Developing syngeneic murine lung cancer cell lines: minimizing host rejection
We hypothesized that passaging tumors through a second animal would allow for additional tumor growth and might select for tumors more likely to grow in a nonself host, however this approach must consider genetic background as murine lung cancer lines developed from mixed backgrounds [12,39]   Howard Li). Accordingly, we generated primary tumors in animals that were > 95% C57BL/6 by SNP analysis and used tumor recipients for passaging that were > 90% C57BL/6 with no SNP mismatches on chromosome 17 where the mouse major histocompatibility locus (MHC) is located. We also sex-matched primary tumors and tumor recipients to reduce the chances of tumor rejection based on sex specific proteins. Despite these steps, greater than 75% (87/113) of primary tumors failed at the first passage while 23% (6/26) of tumors that successfully completed a first passage ultimately gave rise to lines capable of forming orthotopic tumors. Because our goal was to produce lines capable of orthotopic growth in an immunocompetent host, we did not assess the impact of creating cell lines as the first step in cell line generation.

Potential utility of syngeneic murine tumors models
The lines described herein are capable of forming both lung and flank tumors in both male and female recipients (not explicitly shown) however there are clear differences in the flank and lung tumor microenvironment and these differences can critically alter immunotherapeutic responses [16]. For monitoring purposes we passaged through flank to allow for tumor amplification and ease ; tumorigenicity in other background strains was not assessed. Our goal was to establish tumorigenicity of these cell lines in the lungs of C57BL/6 hosts; future investigators will have to optimize experimental conditions with respect to the number of cells injected and the timing of experimental endpoints that balance primary tumor formation and metastases development.
Although we did not directly compare the responses of the novel cell lines to immunotherapy we did find that IFNγ treatment increases PD-L1 mRNA expression in CMT167, Y856, X577, E889, and X381 cells (Additional file 1: Figure  S1); this characteristic is associated with sensitivity to anti-PD-1 treatment in vivo [40]. That LLC and X911 cells fail to respond to INFγ stimulation (and that Y143 cells are equivocal) illustrates how having a broader array of cell lines to test in vivo potentially increases the generalizability of a given observation and also allows investigators to explore mechanistic differences underpinning a specific characteristic. In addition, as these lines have defined oncogenic drivers and retain responsiveness to inhibition of these drivers (at least in the case of the EML4-ALK line), this sets the stage for experiments combining small molecule inhibitor and immunotherapeutic approaches which had previosly been beyond the scope of a typical orthotopic experiment.

Conclusions
We produced six novel murine lung cancer cell lines capable of orthotopic tumor formation in syngeneic immunocompetent animals. These lines will be invaluable for preclinical studies of small molecule inhibitor and immunotherapy combinatorial approaches. Our methods provide a broader road map for the development of additional murine cancer cell lines capable of orthotopic tumor formation in immunocompetent hosts.