Applicability of Stable Murine Cell Lines for Melanoma Research

Abstract

The rates of malignant melanoma are on the rise and, in some populations, lifetime incidence is as high as 1 in 50 individuals. These frequencies are especially concerning given that the current 5-year survival rate of patients with late-stage melanoma is only 15%. While several treatment options have shown promise in the clinic, resistance to therapeutic drugs is common in many patients, emphasizing the need to develop more effective treatment options and a better understanding of the molecular mechanisms driving melanoma development and drug resistance. Many studies seek to develop immune-based strategies to complement current treatment options and increase efficacy; however, the reliance of human melanoma cell lines on xenografts in immune-compromised mice makes immune-based studies difficult. This review discusses available mouse models of melanoma with the common BRAF^V600E mutation, describes a protocol devised to generate syngeneic murine cell lines from one of these mouse models, and highlights potential applications for the use of these cell lines in biotechnology research.

Mouse Models of BRAF^V600E Melanoma

Invasive melanoma is often associated with a mutation in the BRAF oncogene,^1,2 with the most common being BRAF^V600E, which is found in 50% of human melanomas. The BRAF^V600E protein promotes melanoma progression by constitutively activating mitogen activated protein kinase (MAPK) signaling cascades^1–5 and subsequent downstream genes.^1–5 BRAF^V600E is involved not only in the initiation and progression of melanoma, but also in tumor maintenance, making it a good drug target. The small molecule inhibitor, vemurafenib (also known as PLX4032 or its sister compound, PLX4720) binds specifically to the V600E mutated version of BRAF, blocking constitutive activation of the MAPK pathway in the tumor cells,⁴ and inducing rapid tumor regression.^3,6–8 However, development of drug resistance is a major clinical problem for patients with BRAF^V600E mutant melanoma, as the majority of patients are refractory to vemurafenib in less than 1 year of starting the therapy.^1,4,8 Therefore, there is a need to understand the molecular and cellular mechanisms underlying the BRAF mutation, and to develop additional or combination therapeutic strategies, such as immune-based treatments to complement vemurafenib.

Human melanoma cell lines have been used successfully for many applications.⁹ Intradermal injections of these cells into immune-compromised mice give rise to vascularized tumors with a human parenchyma and murine stroma,¹⁰ making them acceptable models for studying treatment sensitivity and metastatic behavior. However, the use of immune-compromised mice limits studies on tumor immunology. Given that spontaneous melanoma are extremely rare in mice,¹⁰ chemically induced and genetically engineered models were generated.¹¹ Several groups engineered mice with melanocyte-specific expression of BRaf^V600E,^12–14 but found that these transgenic mice develop benign melanocytic hyperplasia in vivo, with evidence of senescence. This senescence leads to a very low frequency of spontaneous melanoma from BRaf^V600E mice, making it an inconsistent model to study malignant melanoma. The cell line SM1 was successfully generated from one of these spontaneous BRaf^V600E melanomas, and genetic analysis revealed the depletion of CDKN2A in these cells.^3,14 While this cell line is syngeneic to fully immune-competent mice in vivo, it shows only partial and variable sensitivity to vemurafenib in vitro,^3,15 suggesting that it may not represent many human melanomas in culture.

Mouse models with tyrosinase-driven mouse melanocyte expression of BRaf^V600E combined with the loss of tumor suppressors, such as Pten, Ink4a, or Ink4a/Arf, consistently generate malignant BRaf^V600E melanoma.^12,13 For example, mice with the genotype, Tyr::CreER;Braf^CA;Pten^{lox4-5/lox4-5,13} which encode conditionally active CreERT2 specifically in melanocytes, develop melanoma with 100% penetrance within 1 month of topical administration of 4-hydroxytamoxifen (4-HT).¹³ Such engineered inducible models have the potential to provide important insights into the molecular events initiating human melanomas, and have been used to test combination drug therapies, such as MEK and mTOR inhibitors.¹³

Although the Tyr::CreER;Braf^CA;Pten^{lox4-5/lox4-5} transgenic mouse model was originally bred on a mixed background, several groups have backcrossed this onto a pure C57BL/6 background. While backcrossing these mice has allowed for in vivo immunologic studies in a syngeneic system,^16–19 the ability to perform both in vivo and in vitro experiments using this syngeneic system would provide additional detailed characterization of critical molecular mechanisms:signal transduction pathways, patterns of gene expression, and interactions with cells found within the tumor microenvironment.⁹ Unfortunately, many investigators have found it difficult to develop stable cell lines from the Tyr::CreER;Braf^CA;Pten^{lox4-5/lox4-5} inducible model. Indeed, cell lines derived from this model showed either limited growth potential in vitro or insensitivity to vemurafenib.¹⁶ Further, in vitro results from these cells differed from in vivo results seen in the corresponding mouse model¹⁶ and with human BRAF^V600E cell lines,^4,5,8 somewhat undermining their usefulness.

Thus, murine BRaf^V600E melanoma cell lines that a) grow readily in cell culture, b) form transplantable tumors in syngeneic mice, c) show sensitivity to vemurafenib, and d) express functionally relevant melanoma antigens would be advantageous for in vitro and in vivo studies.

Generation and Characterization of Murine BRaf^V600E Melanoma Cell Lines

Figure 1. Generation of D4M cell lines. 1) BRaf/Pten tumors were induced with 4-HT. 2) Tumors were digested into single cells, and 3) intradermally injected into NSG host mice either directly, or following culture in vitro (<2 weeks). 4) Resulting tumors were digested into single cells and placed in culture in DMEM/F-12 advanced medium + 5% serum. 5) Stable cell lines were termed D4M cell lines. Figure modified from Jenkins et al. 2014 (ref. 20).

Similar to other studies, we backcrossed the transgenic mouse model, Tyr::CreER;Braf^CA;Pten^lox/lox¹³ (a gift from M. Bosenberg) to C57BL/6 (B6) mice to achieve >98% B6 DNA (we refer to these backcrossed mice as BRaf/Pten mice). Primary tumors arising from 4-HT administration in these BRaf/Pten mice were excised and dissociated into single cell suspensions with bacterial collagenase for 1 hour. However, when these primary melanoma cells were placed directly in culture, they grew slowly and were relatively insensitive to vemurafenib, similar to the results of other investigators.^3,13,15,16 With protocol optimization, we found that a critical step in generating cell lines from BRaf/Pten mice was to passage the single cell suspension of tumor cells through immune-compromised NOD/SCID/γ chain^null (NSG) mice (Figure 1). By dissociating the tumors arising from intradermal injections of BRaf/ Pten cells into NSG mice, we were able to routinely establish stable cell lines, from both male and female BRaf/Pten tumors, that grew well in vitro.²⁰ We have termed these stable lines Dartmouth Murine Mutant Malignant Melanoma (D4M) cells.

After establishing a protocol and generating multiple D4M cell lines from different BRaf/Pten primary tumors, we performed characterization studies on several D4M cell lines to confirm that this in vitro model appropriately represents the in vivo biology of BRaf/ Pten tumors. Specifically, cells were assessed for a) in vitro growth, b) in vivo growth in immune-compromised and immune-competent hosts, and c) expression of melanoma-specific antigens.²⁰ These cellular characteristics were analyzed in either the absence or presence of vemurafenib. Given that the V600E BRAF mutation causes constitutive activation of MAPK signaling in human and mouse BRaf^V600E tumors,¹³ we first evaluated downstream phosphorylation of ERK in D4M cell lines. We confirmed that all D4M cell lines had high phERK levels compared to the BRafWT murine melanoma cell line, B16, and that phERK levels were reduced by vemurafenib treatment,²⁰ a response comparable to human BRAF^V600E cell lines.²¹

In vitro Growth

Growth curves demonstrated rapid proliferation of D4M cells in vitro. Importantly, D4M cell lines were able to survive, albeit with more limited growth, in serum-free medium for at least 4 days, thus permitting experiments that require these conditions, such as investigating secreted factors in the medium. Growth rates of D4M cells cultured in serum-containing medium decreased with vemurafenib treatment, in a primarily cytostatic manner, similar to those seen with human BRAF^V600E cell lines.^4,5

In vivo Growth

Intradermal injections into NSG and B6 mice demonstrated that D4M cell lines are highly tumorigenic in both immune-compromised and syngeneic hosts.²⁰ In fact, we saw no differences in growth kinetics or gross tumor morphology in tumors arising in these different hosts. We next performed intradermal injections into B6 mice, waited until the tumors measured 5 mm in diameter, and then fed the mice either control chow or chow containing vemurafenib. Mice fed vemurafenib chow had significantly decreased tumor growth and decreased phERK levels in the tumor tissue. These results confirmed the sensitivity of D4M cells to vemurafenib both in vitro and in vivo.

Expression of Melanoma-specific Markers

Melanocyte differentiation antigens (MDAs), which are often expressed by human melanoma cells and recognized by tumor-infiltrating immune cells,²² make potentially useful biomarkers for murine melanoma. Therefore, to further verify that D4M cell lines have melanoma characteristics, we examined expression of several murine MDAs, including Pmel (also referred to as gp100), Tyrosinase, Mart-1, and Tryp-1. Although levels of these markers were relatively low in D4M cells in vitro, when D4M cells were intradermally injected into mice, the excised tumors had high levels of all markers, regardless of how many times they were injected and recultured.²⁰ These results indicate that D4M cells maintain their ability to express MDAs, and that their response to factors in the host tumor microenvironment increases MDA levels.

Studies with human BRAF^V600E cell lines in vitro have shown that vemurafenib can increase expression of MDAs,^6,23,24 such as PMEL. Likewise, we found that treatment of D4M cells with vemurafenib increased expression of MDAs in vitro, particularly the expression of Pmel. To analyze whether this increase in Pmel was functionally relevant, we used a cytotoxicity assay with murine Pmel TCR transgenic T cells. We found that activated Pmel T cells were able to recognize and lyse the vemurafenib-treated D4M cells much more efficiently than the control-treated D4M cells.²⁰ These results demonstrate that D4M cells express Pmel in a functionally relevant manner.

Taken together, these characterization results are consistent with human BRAF^V600E melanoma cell lines, and suggest that D4M cells recapitulate the human disease. Additional characterization of these cells is ongoing within our group. Specifically, passaging BRaf/Pten cells through NSG mice appears to be selecting for a specific cell type in the heterogeneous primary tumor population. Comparisons between the primary BRaf/Pten cells and stable D4M cell lines may offer insight into mechanisms behind this selection process, and, perhaps, a better understanding of melanoma progression and maintenance. We also find that intradermal injections of D4M cell lines form no gross metastases in the lungs or lymph nodes of either NSG or B6 hosts. One study found that modulating β-catenin levels in the mixed genetic background Tyr::CreER;Braf^CA;Pten^lox/lox melanoma mouse model could mediate metastasis to lymph nodes and lungs.²⁵ Similarly, direct genetic modification of the syngeneic D4M cell lines with genes known to be involved in melanoma metastasis may increase the metastatic potential of D4M cells and increase the applicability of these cells in studies that seek to affect metastasis using immunotherapeutic approaches.

Potential Applications

In summary, characterization of D4M cells demonstrates the unique ability of these cells to correlate in vitro studies on molecular mechanisms of melanoma with in vivo investigations on pathology and immunology, making them a useful model for studying malignant melanoma in many applications. Following are a few examples of the potential applications for D4M cells in melanoma research, but they are by no means limited to these. Because D4M cells grow easily in culture, they can be used for in vitro studies looking at cellular and molecular mechanisms or for genetic manipulation experiments, as they transfect easily. Unlike human cell line xenografts, D4M cells are transplantable in syngeneic host mice, allowing for studies on immunotherapy and the role of the tumor microenvironment. The sensitivity of D4M cells to vemurafenib both in vitro and in vivo offers investigators the ability to test large cohorts of combination drug therapies. Finally, since D4M cells express at least one functionally relevant melanoma-specific antigen, they could be used to study potential targeted immunotherapy.

Human BRAF^V600E melanoma cell lines are relatively heterogeneous. Therefore, having multiple murine BRaf^V600E cell lines available is an important resource for the scientific community. The SM1 cell line, derived from a spontaneous tumor in a BRaf^V600E transgenic mouse,³ has been a useful model in supporting the therapeutic potential of combining BRAF inhibitors with immunotherapy.¹⁵ Similarly, the recent development of the BP cell line was advantageous in exhibiting synergy between combined BRAF inhibitor therapy and immune checkpoint blockade (anti-PD-1 or anti-PD-L1).¹⁸ To date, over 20 investigators have requested D4M cells for their research, indicating that these cell lines likewise represent an important resource for the scientific community worldwide.

Acknowledgement

The author wishes to thank Dr. Constance Brinckerhoff and Dr. David Mullins for their mentorship and help with the D4M project, and other colleagues at the Geisel School of Medicine at Dartmouth College for their support in this work.

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Author Biography

Molly Jenkins, PhD, is a Postdoctoral Research Fellow at the Geisel School of Medicine at Dartmouth College in Lebanon, NH. She received her Masters of Science in 2009 and PhD in Biomedical Sciences in 2011 from the University of Maine, Orono.