Targeting BRAF in an Inducible Murine Model of Melanoma
2012; Elsevier BV; Volume: 181; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2012.06.002
ISSN1525-2191
AutoresAnna I. Hooijkaas, Jules Gadiot, Martin van der Valk, Wolter J. Mooi, Christian U. Blank,
Tópico(s)Quinazolinone synthesis and applications
ResumoThe MAP kinase and PI3 kinase pathways have been identified as the most common pathways that mediate oncogenic transformation in melanoma, and the majority of compounds developed for melanoma treatment target one or the other of these pathways. In addition to such targeted therapies, immunotherapeutic approaches have shown promising results. A combination of these two treatment modalities could potentially result in further improvement of treatment outcome. To preclinically identify efficient treatment combinations and to optimize therapy protocols in terms of sequence and timing, mouse models will be required. We have crossed and characterized the Tyr::CreERT2;PTENF−/−;BRAFF-V600E/+ inducible melanoma model on a C57BL/6J background. Tumors from this model harbor the BRAFV600E mutation and are PTEN-deficient, making them highly suitable for the testing of targeted therapies. Furthermore, we crossed the model onto this specific background for use in immunotherapy studies, because most experiments in this field have been performed in C57BL/6J mice. Selective inhibition of BRAFV600E by PLX4720 treatment of melanoma-bearing mice resulted in a strong decrease of tumor outgrowth. Furthermore, the inducible melanomas had immune cell infiltrates similar to those found in human melanoma, and tumor-infiltrating lymphocytes could be cultured from these tumors. Our data indicate that the C57BL/6J Tyr::CreERT2;PTENF−/−;BRAFF-V600E/+ melanoma model could be used as a standard model in which targeted and immunotherapy combinations can be tested in a high-throughput manner. The MAP kinase and PI3 kinase pathways have been identified as the most common pathways that mediate oncogenic transformation in melanoma, and the majority of compounds developed for melanoma treatment target one or the other of these pathways. In addition to such targeted therapies, immunotherapeutic approaches have shown promising results. A combination of these two treatment modalities could potentially result in further improvement of treatment outcome. To preclinically identify efficient treatment combinations and to optimize therapy protocols in terms of sequence and timing, mouse models will be required. We have crossed and characterized the Tyr::CreERT2;PTENF−/−;BRAFF-V600E/+ inducible melanoma model on a C57BL/6J background. Tumors from this model harbor the BRAFV600E mutation and are PTEN-deficient, making them highly suitable for the testing of targeted therapies. Furthermore, we crossed the model onto this specific background for use in immunotherapy studies, because most experiments in this field have been performed in C57BL/6J mice. Selective inhibition of BRAFV600E by PLX4720 treatment of melanoma-bearing mice resulted in a strong decrease of tumor outgrowth. Furthermore, the inducible melanomas had immune cell infiltrates similar to those found in human melanoma, and tumor-infiltrating lymphocytes could be cultured from these tumors. Our data indicate that the C57BL/6J Tyr::CreERT2;PTENF−/−;BRAFF-V600E/+ melanoma model could be used as a standard model in which targeted and immunotherapy combinations can be tested in a high-throughput manner. The incidence and mortality of melanoma have steadily risen over the past two decades.1Chapman P.B. Hauschild A. Robert C. Haanen J.B. Ascierto P. Larkin J. Dummer R. Garbe C. Testori A. Maio M. Hogg D. Lorigan P. Lebbe C. Jouary T. Schadendorf D. Ribas A. O'Day S.J. Sosman J.A. Kirkwood J.M. Eggermont A.M. Dreno B. Nolop K. Li J. Nelson B. Hou J. Lee R.J. Flaherty K.T. McArthur G.A. BRIM-3 Study GroupImproved survival with vemurafenib in melanoma with BRAF V600E mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6311) Google Scholar, 2Robert C. Thomas L. Bondarenko I. O'Day S. M D J.W. Garbe C. Lebbe C. Baurain J.F. Testori A. Grob J.J. Davidson N. Richards J. Maio M. Hauschild A. Miller Jr, W.H. Gascon P. Lotem M. Harmankaya K. Ibrahim R. Francis S. Chen T.T. Humphrey R. Hoos A. Wolchok J.D. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma.N Engl J Med. 2011; 364: 2517-2526Crossref PubMed Scopus (3690) Google Scholar However, the mortality rate will most likely change within coming years, because of the development of novel methods to modulate immune cell activation and the development of targeted therapies directed against mutated proteins involved in melanoma.1Chapman P.B. Hauschild A. Robert C. Haanen J.B. Ascierto P. Larkin J. Dummer R. Garbe C. Testori A. Maio M. Hogg D. Lorigan P. Lebbe C. Jouary T. Schadendorf D. Ribas A. O'Day S.J. Sosman J.A. Kirkwood J.M. Eggermont A.M. Dreno B. Nolop K. Li J. Nelson B. Hou J. Lee R.J. Flaherty K.T. McArthur G.A. BRIM-3 Study GroupImproved survival with vemurafenib in melanoma with BRAF V600E mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6311) Google Scholar, 2Robert C. Thomas L. Bondarenko I. O'Day S. M D J.W. Garbe C. Lebbe C. Baurain J.F. Testori A. Grob J.J. Davidson N. Richards J. Maio M. Hauschild A. Miller Jr, W.H. Gascon P. Lotem M. Harmankaya K. Ibrahim R. Francis S. Chen T.T. Humphrey R. Hoos A. Wolchok J.D. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma.N Engl J Med. 2011; 364: 2517-2526Crossref PubMed Scopus (3690) Google Scholar, 3Blank C.U. Hooijkaas A.I. Haanen J.B. Schumacher T.N. Combination of targeted therapy and immunotherapy in melanoma.Cancer Immunol Immunother. 2011; 60: 1359-1371Crossref PubMed Scopus (39) Google Scholar, 4Chappell W.H. Steelman L.S. Long J.M. Kempf R.C. Abrams S.L. Franklin R.A. Basecke J. Stivala F. Donia M. Fagone P. Malaponte G. Mazzarino M.C. Nicoletti F. Libra M. Maksimovic-Ivanic D. Mijatovic S. Montalto G. Cervello M. Laidler P. Milella M. Tafuri A. Bonati A. Evangelisti C. Cocco L. Martelli A.M. McCubrey J.A. Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR inhibitors: rationale and importance to inhibiting these pathways in human health.Oncotarget. 2011; 2: 135-164Crossref PubMed Scopus (491) Google Scholar These efforts have led to two phase-3-approved molecules, representative of a broad range of further upcoming therapeutic options in melanoma.1Chapman P.B. Hauschild A. Robert C. Haanen J.B. Ascierto P. Larkin J. Dummer R. Garbe C. Testori A. Maio M. Hogg D. Lorigan P. Lebbe C. Jouary T. Schadendorf D. Ribas A. O'Day S.J. Sosman J.A. Kirkwood J.M. Eggermont A.M. Dreno B. Nolop K. Li J. Nelson B. Hou J. Lee R.J. Flaherty K.T. McArthur G.A. BRIM-3 Study GroupImproved survival with vemurafenib in melanoma with BRAF V600E mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6311) Google Scholar, 2Robert C. Thomas L. Bondarenko I. O'Day S. M D J.W. Garbe C. Lebbe C. Baurain J.F. Testori A. Grob J.J. Davidson N. Richards J. Maio M. Hauschild A. Miller Jr, W.H. Gascon P. Lotem M. Harmankaya K. Ibrahim R. Francis S. Chen T.T. Humphrey R. Hoos A. Wolchok J.D. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma.N Engl J Med. 2011; 364: 2517-2526Crossref PubMed Scopus (3690) Google Scholar, 5Hodi F.S. O'Day S.J. McDermott D.F. Weber R.W. Sosman J.A. Haanen J.B. Gonzalez R. Robert C. Schadendorf D. Hassel J.C. Akerley W. van den Eertwegh A.J. Lutzky J. Lorigan P. Vaubel J.M. Linette G.P. Hogg D. Ottensmeier C.H. Lebbé C. Peschel C. Quirt I. Clark J.I. Wolchok J.D. Weber J.S. Tian J. Yellin M.J. Nichol G.M. Hoos A. Urba W.J. Improved survival with ipilimumab in patients with metastatic melanoma [Erratum appeared in N Engl J Med 2010, 363:1290].N Engl J Med. 2010; 363: 711-723Crossref PubMed Scopus (11783) Google Scholar First, ipilimumab, a phase-3-approved monoclonal antibody directed against cytotoxic T-lymphocyte antigen-4 (CTLA-4), represents the immunotherapeutic approach to melanoma treatment.2Robert C. Thomas L. Bondarenko I. O'Day S. M D J.W. Garbe C. Lebbe C. Baurain J.F. Testori A. Grob J.J. Davidson N. Richards J. Maio M. Hauschild A. Miller Jr, W.H. Gascon P. Lotem M. Harmankaya K. Ibrahim R. Francis S. Chen T.T. Humphrey R. Hoos A. Wolchok J.D. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma.N Engl J Med. 2011; 364: 2517-2526Crossref PubMed Scopus (3690) Google Scholar, 5Hodi F.S. O'Day S.J. McDermott D.F. Weber R.W. Sosman J.A. Haanen J.B. Gonzalez R. Robert C. Schadendorf D. Hassel J.C. Akerley W. van den Eertwegh A.J. Lutzky J. Lorigan P. Vaubel J.M. Linette G.P. Hogg D. Ottensmeier C.H. Lebbé C. Peschel C. Quirt I. Clark J.I. Wolchok J.D. Weber J.S. Tian J. Yellin M.J. Nichol G.M. Hoos A. Urba W.J. Improved survival with ipilimumab in patients with metastatic melanoma [Erratum appeared in N Engl J Med 2010, 363:1290].N Engl J Med. 2010; 363: 711-723Crossref PubMed Scopus (11783) Google Scholar Other promising immunotherapeutic approaches are monoclonal antibodies targeting the coinhibitory PD-1/PD-L1 pathway or adoptive cell therapies using tumor infiltrating lymphocytes (TILs) or T-cell receptor gene-modified T cells.6Blank C. Kuball J. Voelkl S. Wiendl H. Becker B. Walter B. Majdic O. Gajewski T.F. Theobald M. Andreesen R. Mackensen A. Blockade of PD-L1 (B7-H1) augments human tumor-specific T cell responses in vitro.Int J Cancer. 2006; 119: 317-327Crossref PubMed Scopus (273) Google Scholar, 7Dudley M.E. Adoptive cell therapy for patients with melanoma.J Cancer. 2011; 2: 360-362Crossref PubMed Scopus (17) Google Scholar Response rates after immunotherapy such as ipilimumab treatment are characteristically low, but the responses tend to be of long duration.8Di Giacomo A.M. Danielli R. Guidoboni M. Calabrò L. Carlucci D. Miracco C. Volterrani L. Mazzei M.A. Biagioli M. Altomonte M. Maio M. Therapeutic efficacy of ipilimumab, an anti-CTLA-4 monoclonal antibody, in patients with metastatic melanoma unresponsive to prior systemic treatments: clinical and immunological evidence from three patient cases.Cancer Immunol Immunother. 2009; 58: 1297-1306Crossref PubMed Scopus (219) Google Scholar, 9O'Day S.J. Maio M. Chiarion-Sileni V. Gajewski T.F. Pehamberger H. Bondarenko I.N. Queirolo P. Lundgren L. Mikhailov S. Roman L. Verschraegen C. Humphrey R. Ibrahim R. de Pril V. Hoos A. Wolchok J.D. Efficacy and safety of ipilimumab monotherapy in patients with pretreated advanced melanoma: a multicenter single-arm phase II study.Ann Oncol. 2010; 21: 1712-1717Crossref PubMed Scopus (452) Google Scholar Second, vemurafenib, a new small molecule specifically inhibiting the mutated BRAFV600E protein, represents the targeted therapy approach of melanoma treatment. Other promising targeted therapy molecules tested in phase 1 and 2 studies are selective inhibitors of MEK, ERK, PI3K, AKT, and mTOR.4Chappell W.H. Steelman L.S. Long J.M. Kempf R.C. Abrams S.L. Franklin R.A. Basecke J. Stivala F. Donia M. Fagone P. Malaponte G. Mazzarino M.C. Nicoletti F. Libra M. Maksimovic-Ivanic D. Mijatovic S. Montalto G. Cervello M. Laidler P. Milella M. Tafuri A. Bonati A. Evangelisti C. Cocco L. Martelli A.M. McCubrey J.A. Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR inhibitors: rationale and importance to inhibiting these pathways in human health.Oncotarget. 2011; 2: 135-164Crossref PubMed Scopus (491) Google Scholar Unlike immunotherapy, targeted therapies such as vemurafenib induce high and fast response rates in melanomas, but progression-free survival tends to be of short duration.1Chapman P.B. Hauschild A. Robert C. Haanen J.B. Ascierto P. Larkin J. Dummer R. Garbe C. Testori A. Maio M. Hogg D. Lorigan P. Lebbe C. Jouary T. Schadendorf D. Ribas A. O'Day S.J. Sosman J.A. Kirkwood J.M. Eggermont A.M. Dreno B. Nolop K. Li J. Nelson B. Hou J. Lee R.J. Flaherty K.T. McArthur G.A. BRIM-3 Study GroupImproved survival with vemurafenib in melanoma with BRAF V600E mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6311) Google Scholar To further improve melanoma treatment, it will be important to reduce the occurrence of tumor escape on treatment and to increase the number of long-term responses. Three types of treatment combinations can be envisioned to achieve this.3Blank C.U. Hooijkaas A.I. Haanen J.B. Schumacher T.N. Combination of targeted therapy and immunotherapy in melanoma.Cancer Immunol Immunother. 2011; 60: 1359-1371Crossref PubMed Scopus (39) Google Scholar First, in-pathway combinations that simultaneously target one signaling pathway by multiple small-molecule inhibitors. Second, cross-pathway combinations that target different signaling pathways at the same time. Third, the combination of multiple small molecule inhibitors with immunotherapeutic approaches. Clinical testing of such large numbers of possible treatment combinations and the optimization of therapy protocols in terms of timing and sequence of treatment would take many years and a great many phase 1/2 studies in melanoma patients. Thus, preclinical testing using a mouse model of human melanoma would be a substantial asset. This murine model should represent genetic lesions commonly found in human melanoma in combination with an intact tumor microenvironment, immune system, and angiogenesis.10Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Abstract Full Text Full Text PDF PubMed Scopus (45922) Google Scholar Most of the murine melanoma models currently used for studying targeted therapies are models in which human tumor cell lines are xenotransplanted into immunodeficient hosts. Such xenotransplantation models mimic advanced metastatic melanoma, but without considering the alterations in angiogenesis and the important roles of the immune system and tumor microenvironment.10Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Abstract Full Text Full Text PDF PubMed Scopus (45922) Google Scholar In light of recent data showing that intact immune systems are relevant for the effect of targeted therapy and can also contribute to tumor escape,11Denardo D.G. Brennan D.J. Rexhepaj E. Ruffell B. Shiao S.L. Madden S.F. Gallagher W.M. Wadhwani N. Keil S.D. Junaid S.A. Rugo H.S. Hwang E.S. Jirström K. West B.L. Coussens L.M. Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy.Cancer Discov. 2011; 1: 54-67Crossref PubMed Scopus (521) Google Scholar, 12Rakhra K. Bachireddy P. Zabuawala T. Zeiser R. Xu L. Kopelman A. Fan A.C. Yang Q. Braunstein L. Crosby E. Ryeom S. Felsher D.W. CD4(+) T cells contribute to the remodeling of the microenvironment required for sustained tumor regression upon oncogene inactivation [Erratum appeared in Cancer Cell 2010, 18:696].Cancer Cell. 2010; 18: 485-498Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar xenotransplantation melanoma models are seen as less suitable for preclinical testing of immune and targeted therapy combination treatment options. Similarly, syngeneic transplantation models, such as the B16 mouse melanoma model, are unsuitable for such purposes, because these tumors not only fail to resemble human melanoma histologically, but also lack the genetic lesions found in human melanoma.13Becker J.C. Houben R. Schrama D. Voigt H. Ugurel S. Reisfeld R.A. Mouse models for melanoma: a personal perspective.Exp Dermatol. 2010; 19: 157-164Crossref PubMed Scopus (65) Google Scholar Consequently, clinicians are currently forced to plan upcoming combination studies of drugs such as vemurafenib and ipilimumab without any guidance from preclinical data with respect to relative dosing or treatment sequence. Although the use of genetically modified mouse models of melanoma for efficient screening of treatment combinations is generally hampered by low tumor incidence and long latency, this was not the case for one model recently described by Dankort et al.14Dankort D. Curley D.P. Cartlidge R.A. Nelson B. Karnezis A.N. Damsky Jr, W.E. You M.J. DePinho R.A. McMahon M. Bosenberg M. Braf(V600E) cooperates with Pten loss to induce metastatic melanoma.Nat Genet. 2009; 41: 544-552Crossref PubMed Scopus (887) Google Scholar This tamoxifen-inducible melanoma model on a mixed genetic background used both the BRAFV600E mutation and deficiency of PTEN for the malignant transformation of melanocytes. The BRAFV600E mutation is found in 40% to 60% of all human melanomas.15Bauer J. Büttner P. Murali R. Okamoto I. Kolaitis N.A. Landi M.T. Scolyer R.A. Bastian B.C. BRAF mutations in cutaneous melanoma are independently associated with age, anatomic site of the primary tumor, and the degree of solar elastosis at the primary tumor site.Pigment Cell Melanoma Res. 2011; 24: 345-351Crossref PubMed Scopus (183) Google Scholar, 16Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8529) Google Scholar This mutation is known to lead to aberrant activity of the mitogen-activated protein kinase (MAP kinase) pathway, but sole presence of the BRAFV600E mutation results in induction of senescence.17Michaloglou C. Vredeveld L.C. Soengas M.S. Denoyelle C. Kuilman T. van der Horst C.M. Majoor D.M. Shay J.W. Mooi W.J. Peeper D.S. BRAFE600-associated senescence-like cell cycle arrest of human naevi.Nature. 2005; 436: 720-724Crossref PubMed Scopus (1727) Google Scholar The additional loss of functional PTEN, which is observed in 55% of melanoma metastases and leads to hyperactivity of the PI3 kinase pathway, proved to be sufficient to facilitate malignant transformation of melanocytes in this inducible melanoma model.18Ibrahim N. Haluska F.G. Molecular pathogenesis of cutaneous melanocytic neoplasms.Annu Rev Pathol. 2009; 4: 551-579Crossref PubMed Scopus (98) Google Scholar, 19Smalley K.S. Understanding melanoma signaling networks as the basis for molecular targeted therapy.J Invest Dermatol. 2010; 130: 28-37Crossref PubMed Scopus (107) Google Scholar Because MAP kinase and PI3 kinase pathway hyperactivation is a common denominator for the majority of melanomas, many recently developed small-molecule inhibitors target proteins in either one of these pathways, and these can easily be preclinically tested in this melanoma model. We aimed in the present study to characterize the transgenic melanoma model on a C57BL/6J genetic background, because most experiments in the immunotherapy field have been performed in C57BL/6J mice.20Gemmell E. Winning T.A. Carter C.L. Ford P.J. Bird P.S. Ashman R.B. Grieco D.A. Seymour G.J. Differences in mouse strain influence leukocyte and immunoglobulin phenotype response to Porphyromonas gingivalis.Oral Microbiol Immunol. 2003; 18: 364-370Crossref PubMed Scopus (15) Google Scholar, 21Kerkar S.P. Sanchez-Perez L. Yang S. Borman Z.A. Muranski P. Ji Y. Chinnasamy D. Kaiser A.D. Hinrichs C.S. Klebanoff C.A. Scott C.D. Gattinoni L. Morgan R.A. Rosenberg S.A. Restifo N.P. Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies.J Immunother. 2011; 34: 343-352Crossref PubMed Scopus (67) Google Scholar, 22Klebanoff C.A. Gattinoni L. Palmer D.C. Muranski P. Ji Y. Hinrichs C.S. Borman Z.A. Kerkar S.P. Scott C.D. Finkelstein S.E. Rosenberg S.A. Restifo N.P. Determinants of successful CD8+ T-cell adoptive immunotherapy for large established tumors in mice.Clin Cancer Res. 2011; 17: 5343-5352Crossref PubMed Scopus (219) Google Scholar The inducible melanoma model thus meets the requirements for preclinical testing of targeted therapies and immunotherapies, singly and in various combinations. To illustrate this, we successfully targeted the BRAFV600E mutation using PLX4720, a counterpart of clinical vemurafenib, in the transgenic melanoma model. All animal experiments were performed in accordance with institutional and national guidelines and were approved by the Experimental Animal Committee of the Netherlands Cancer Institute. The inducible mouse melanoma model was obtained by crossing the C57BL/6J Tyr::CreERT2 (homo- or heterozygotic), C57BL/6J PTENF−/− (homozygotic), and mixed-background BRAF F-V600E/+ (heterozygotic) mouse strains.23Dankort D. Filenova E. Collado M. Serrano M. Jones K. McMahon M. A new mouse model to explore the initiation, progression, and therapy of BRAFV600E-induced lung tumors.Genes Dev. 2007; 21: 379-384Crossref PubMed Scopus (383) Google Scholar, 24Marino S. Krimpenfort P. Leung C. van der Korput H.A. Trapman J. Camenisch I. Berns A. Brandner S. PTEN is essential for cell migration but not for fate determination and tumourigenesis in the cerebellum.Development. 2002; 129: 3513-3522PubMed Google Scholar, 25Yajima I. Belloir E. Bourgeois Y. Kumasaka M. Delmas V. Larue L. Spatiotemporal gene control by the Cre-ERT2 system in melanocytes.Genesis. 2006; 44: 34-43Crossref PubMed Scopus (66) Google Scholar These three strains were kindly provided by L. Larue (Institute Curie, France), P. Krimpenfort (NKI-AVL, Netherlands), and M. McMahon (University of California, San Francisco), respectively. The F1 offspring of the Tyr::CreERT2;PTENF−/−;BRAFF-V600E/+ mice were backcrossed to C57BL/6J mice for an additional six generations. The genetic background of the resulting mice was determined by the Jackson Laboratory (JAX Mouse Diversity Genotyping Array Service; Bar Harbor, ME), using high-density, genome-wide profiling of single nucleotide polymorphisms (SNPs). Analysis using tissue samples from three individual mice indicated that >97% of the analyzed SNPs matched the C57BL/6J predicted SNP reference. Tumors were induced on the skin of the melanoma model mice as described previously.14Dankort D. Curley D.P. Cartlidge R.A. Nelson B. Karnezis A.N. Damsky Jr, W.E. You M.J. DePinho R.A. McMahon M. Bosenberg M. Braf(V600E) cooperates with Pten loss to induce metastatic melanoma.Nat Genet. 2009; 41: 544-552Crossref PubMed Scopus (887) Google Scholar In brief, 2 μL of 5 mmol/L 4-hydroxytamoxifen ≥70% Z isomer (remainder primarily E-isomer) (Sigma-Aldrich, St. Louis, MO) dissolved in pure dimethyl sulfoxide (Sigma-Aldrich) was topically applied for 5 minutes to the shaven right flank of 4- to 10-week-old mice for three consecutive days. Tumor outgrowth was typically monitored twice weekly, from 28 days after tumor induction, by digital photography of the tumor, including a size reference. Tumor size was then analyzed in two dimensions using ImageJ software version 1.45S (NIH, Bethesda, MD). A 10 × 10-mm tumor was removed from a C57BL/6J Tyr::CreERT2;PTENF−/−;BRAFF-V600E/+ mouse, and minced tumor fragments were digested in serum-free Dulbecco's modified Eagle's medium containing 3 mg/mL collagenase type A (Roche, Basel, Switzerland) and 1.5 mg/mL porcine pancreatic trypsin (BD Biosciences, San Jose, CA) for 1 hour at room temperature. Subsequently, 9-week-old male C57BL/6J mice were subcutaneously injected with 1 × 106 cells from this digest, and tumor growth was monitored over time by caliper measurements. Melanomas were induced in the Tyr::CreERT2;PTENF−/−;BRAFF-V600E/+ mice on day 0, as described above. On day 31, treatment was started either by administering 20 or 100 mg/kg PLX4720 dissolved in 1% carboxymethylcellulose (Sigma-Aldrich) and 10% dimethyl sulfoxide (Sigma-Aldrich) once daily by oral gavage for 21 days or by switching the food provision of the mice to either a chow diet containing 417 mg/kg PLX4720 (treatment arm) or control chow containing no compound (control arm). Both soluble PLX4720 and the chow version were provided by Plexxikon (Berkeley, CA). At different time points during the treatment, plasma samples were taken from the mice and sent to Integrated Analytical Solutions (Berkeley, CA) for determination of PLX4720 drug plasma levels. Immunohistochemical staining of formalin-fixed, paraffin-embedded tumor samples was performed as described previously.26Gadiot J. Hooijkaas A.I. Kaiser A.D. van T.H. van B.H. Blank C. Overall survival and PD-L1 expression in metastasized malignant melanoma.Cancer. 2011; 117: 2192-2201Crossref PubMed Scopus (201) Google Scholar For staining, the following primary antibodies were used: anti-TRP2 clone D-18, 1:400 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Melan-A clone PNLA, 1:50 (Abcam, Cambridge, UK), anti-GP100 clone HMB45, 1:1000 (Pierce; Thermo Fisher Scientific, Rockford, IL), anti-CD3 clone SP7, 1:600 (Pierce; Thermo Fisher Scientific), and anti-CD45 clone 30-F11, 1:400 (BD Biosciences, Franklin Lakes, NJ). Romulin 3-amino-9-ethylcarbazole (BioCare Medical, Concord, CA) was used to visualize positive staining, followed by a hematoxylin counterstain. Tumors (and in some cases draining lymph nodes as well) were removed from the animals directly after euthanasia. Subsequently, single-cell suspensions from the collected tumor tissues were obtained by continuous slicing of the tumor, followed by pressing the material through a 70-μm filter (BD Biosciences). The obtained single-cell suspension was then stained with the following fluorochrome-labeled antibodies from BD Biosciences or from eBioscience (San Diego, CA): anti-CD3 clone 17A2, anti-CD4 clone GK1.5, anti-CD8 clone 53-6.7, anti-NK1.1 clone PK136, anti-CD11b clone M1/70, anti-CD11c clone N418, anti-B220 clone RA3-6B2, anti-CD19 clone 1D3, anti-IA/IE clone M5/114.15.2, anti-Gr-1 clone RB6-8C5, anti-F4/80 clone BM8, anti-FoxP3 clone FJK-165, anti-CD25 clone PC61, anti-CD45 clone 104, anti-PD-1 clone J43, and its isotype control anti-Armenian hamster IgG. Intracellular staining of the FoxP3 molecule was performed according to the manufacturer's protocol (eBioscience). The standardized fluorescence intensity (SFI) was calculated for PD-1 expression by dividing the mean fluorescence intensity (MFI) of the PD-1 staining by the MFI of the isotype. Samples were acquired on a FACSCalibur (BD Biosciences) fluorescence-activated cell sorting system and were analyzed using FlowJo software version 7.6.5 (Tree Star, Ashland, OR). Propidium iodide (eBioscience) was added during sample acquisition to identify dead cells; cells positive for propidium iodide were discarded. Tumor infiltrating lymphocytes were cultured from tumor digests and tumor fragments according to standard protocols used for human TILs.27Besser M.J. Schallmach E. Oved K. Treves A.J. Markel G. Reiter Y. Schachter J. Modifying interleukin-2 concentrations during culture improves function of T cells for adoptive immunotherapy.Cytotherapy. 2009; 11: 206-217Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 28Dudley M.E. Wunderlich J.R. Shelton T.E. Even J. Rosenberg S.A. Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients.J Immunother. 2003; 26: 332-342Crossref PubMed Scopus (546) Google Scholar To obtain single cells from the tumor, minced tumor fragments were digested in medium containing 100 U/mL penicillin (Roche), 100 μg/mL streptomycin (Roche), 1 mg/mL Clostridium histolyticum collagenase (Sigma-Aldrich), 30 U/mL DNase-1 (Sigma-Aldrich), and 100 μg/mL sheep testis type V hyaluronidase (Sigma-Aldrich) for 4 hours at room temperature. After washing of the filtered digest twice in Hank's balanced salt solution (Life Technologies-Invitrogen, Carlsbad, CA), the cells were counted, and 100,000 cells/mL were seeded in a tissue culture treated 24-well plate (BD Biosciences). To culture TILs from fragments, the tumor was cut into pieces of <1 mm3; subsequently, one tumor piece was plated per well in a 24-well tissue culture treated plate. Both tumor digests and tumor fragments were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 1× GlutaMAX-1 (Invitrogen), 1× modified Eagle's medium nonessential amino acids (MEM NEAA; Invitrogen), 1× sodium pyruvate (Invitrogen), 100 U/mL penicillin (Roche), 100 μg/mL streptomycin (Roche), 5 μg/mL β-mercaptoethanol, and 300 IU/mL ProLeukin IL-2 (Novartis, Basel, Switzerland). Medium was refreshed twice weekly. Tumor cell lines were cultured from tumor fragments obtained as described above. The tumor fragments were cultured in 24-well tissue culture treated plates (1 fragment per well) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 100 U/mL penicillin (Roche), and 100 μg/mL streptomycin (Roche). Medium was refreshed weekly. Sensitivity for BRAFV600E inhibition was determined for the murine BRAFV600E/PTEN−/− cell line, the murine B16-F10 (ATCC, Manassas, VA) cell line (BRAFV600E-negative), and a human BRAFV600E-positive melanoma cell line. At day 0, 2500 tumor cells per well were plated in a 96-well flat-bottom tissue culture treated plate (BD Biosciences). Subsequently, a dose range of the selective BRAF inhibitor PLX4720 (Plexxikon), dimethyl sulfoxide control (Sigma-Aldrich), or the toxic agent phenylarsine oxide (PAO; Sigma-Aldrich) was added to the indicated wells in triplicate; doses ranged from 80 to 0.19 μmol/L in twofold dilution steps. Cells were left at 37°C for 72 hours before the total metabolic activity per well was measured using CellTiter-Blue reagent (Promega, Madison, WI), according to the manufacturer's instructions. BRAFV600E/PTEN−/− and B16-F10 cells were cultured in the presence of a dose range (0, 0.75, 1.5, 3, 6, 12.5, and 25 μmol/L) of PLX4720 for 72 hours. Cells were harvested and lysed in 25 mmol/L Tris-HCl (pH 7.6), 1% Nonidet P-40, 150 mmol/L NaCl, 1% sodium deoxycholate, 0.1% SDS, 1 mmol/L phenylmethylsulfonyl fluoride, Roche complete protease inhibitors, and Roche complete phosphatase inhibitors. Clear cell lysates were acquired by centrifugation for 15 minutes at 13,000 × g and subsequent sonication of the samples for 15 minutes. The protein content of each sample was then determined using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA), according to the manufacturer's instructions. Samples containing 30 μg of proteins were prepared in NuPAGE sample buffer (Invitrogen) supplemented with 0.1 mol/L dithiothreitol and were heated for 10 minutes at 95°C before being loaded onto a NuPAGE 4%
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