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. 2015 May;135(5):1405-1414.
doi: 10.1038/jid.2014.411. Epub 2014 Sep 18.

Mechanisms of chemical cooperative carcinogenesis by epidermal Langerhans cells

Julia M Lewis  1 , Christina D Bürgler  1 , Juliet A Fraser  1 , Haihui Liao  1 , Kseniya Golubets  1 , Cynthia L Kucher  2 , Peter Y Zhao  1 , Renata B Filler  1 , Robert E Tigelaar  1 , Michael Girardi  3
Affiliations

Mechanisms of chemical cooperative carcinogenesis by epidermal Langerhans cells

Julia M Lewis et al. J Invest Dermatol. 2015 May.

Abstract

Cutaneous squamous cell carcinoma (SCC) is the most prevalent invasive malignancy with metastatic potential. The epidermis is exposed to a variety of environmental DNA-damaging chemicals, principal among which are polyaromatic hydrocarbons (PAHs) ubiquitous in the environment, tobacco smoke, and broiled meats. Langerhans cells (LCs) comprise a network of dendritic cells situated adjacent to basal, suprabasal, and follicular infundibular keratinocytes that when mutated can give rise to SCC, and LC-intact mice are markedly more susceptible than LC-deficient mice to chemical carcinogenesis provoked by initiation with the model PAH, 7,12-dimethylbenz[a]anthracene (DMBA). LCs rapidly internalize and accumulate DMBA as numerous membrane-independent cytoplasmic foci. Repopulation of LC-deficient mice using fetal liver LC-precursors restores DMBA-induced tumor susceptibility. LC expression of p450 enzyme CYP1B1 is required for maximal rapid induction of DNA-damage within adjacent keratinocytes and their efficient neoplastic transformation; however, effects of tumor progression also attributable to the presence of LC were revealed as CYP1B1 independent. Thus, LCs make multifaceted contributions to cutaneous carcinogenesis, including via the handling and metabolism of chemical mutagens. Such findings suggest a cooperative carcinogenesis role for myeloid-derived cells resident within cancer susceptible epithelial tissues principally by influencing early events in malignant transformation.

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Conflict of interest statement

Conflict of Interest

The authors state no conflict of interest.

Figures

Figure 1
Figure 1. LC facilitate DMBA-initiated cutaneous carcinogenesis and display phenotypic and morphologic changes in response to DMBA exposure
(a)Carcinogenesis initiated in LC-intact (NLC) and LC-deficient (DTA) mice by application of DMBA (400 nmoles) followed by twice weekly TPA (20 nmoles, n=13–15 mice/group, *** P ≤ 0.001). (b) XS106 was treated with 64μM DMBA and changes in gene expression monitored by quantitative real-time PCR, * P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001. (c) FVB/N dorsal bodywall was treated with vehicle (acetone) or 400 nmoles DMBA, epidermal cell suspensions prepared 24 hours later and stained for flow cytometric analysis. Population shown is gated on CD45+ CD207+ cells. LC from untreated (not shown) and vehicle treated mice had similar CD86 expression (ΔMFI = 20.4 vs 17.3; P = n.s.). LC CD86 expression increased following DMBA treatment (ΔMFI = 54.4, P = 0.0013 vs vehicle, n=3 mice/group). (d,e) FVB/N dorsal ears were treated with 35 nmoles DMBA, epidermal sheets prepared 24 hours later and stained with CD207 (red) + MHC II (green). LC were identified and their shape factor (circularity ranging from 0–1, circle = 1. Mean ± SE untreated 0.1825 ± 0.0019, DMBA treated 0.2254 ± 0.0028, P < 0.0001) measured using Volocity 6.2, n=3 mice/group. Scalebar = 10μm.
Figure 2
Figure 2. LC rapidly internalize DMBA as intracellular foci
(a) DMBA (64μM) was added to the LC cell line, XS106, or isolated LC and DMBA fluorescence followed using two-photon microscopy and time-series images collected every 2 minutes (XS106, Supplementary Movie S1 online) or 2.5 minutes (LC, Supplementary Movie S2 online) over 1 hour. DMBA foci within individual cells (n=148 XS106, n=81 LC) were identified and quantitated over the first 25 minutes using ImageJ. (b) Individual foci identified within XS106 during the first 10 minutes of DMBA exposure were measured and followed using ImageJ. (c) Compressed z-stack of isolated LC (FITC-MHC II, green) following 1 hour DMBA (blue) exposure. Single slices of z-stack (Supplementary Figure S1 online) suggest nuclear exclusion of DMBA foci. (d) XS106 cells were labeled with FITC-MHC II (green) and the nuclear dye CyTRAK Orange (red) then exposed to DMBA (blue) for 1 hour. DMBA foci were excluded from the nucleus. (e) XS106 were labeled with the membrane dye PKH26 (red) then exposed to DMBA (blue) for 1 hour. (f) Isolated LC were exposed to DMBA for 1 hour, then washed and cultured for an additional 23 hours in the absence of DMBA (Supplementary Movie S3 online). At this time, 42% of LC retained at least 1 DMBA focus, exemplified in the single slice of the z-stack shown.
Figure 3
Figure 3. Embryonically derived LC-precursors repopulate LC-deficient DTA mice
(a) Cell suspensions prepared from w.t. E10.5 yolk sac or E12.5 liver were injected i.p. into newborn LC-deficient DTA recipients. Three or eight weeks later, flow cytometric analysis of epidermal cell suspensions prepared from recipients as well as NLC and DTA controls were used to determine the extent of LC repopulation. (b) Immunofluorescent staining of an epidermal sheet prepared from a DTA recipient 3 weeks following E12.5 liver transfer show expanding groups of LC (CD207, green). Scalebar = 500 μm. Inset scalebar = 100 μm. (c) E12.5 liver suspensions prepared from w.t. or CYP1B1−/− donors were injected i.p. into recipients of indicated ages. Epidermal cell suspensions prepared 8 weeks later and analyzed by flow cytometry show decreasing LC repopulation with increasing recipient age at time of transfer. No repopulation was detectable when recipients were 5 weeks old at transfer. (d) Flow cytometric analysis of epidermal cell suspensions prepared from bodywall skin of 8 week old NLC, DTA and DTA recipients of E12.5 liver. Upper panels are gated on total epidermal cells; lower panels are gated on CD45+ cells. (e) Epidermal sheets prepared from bodywall skin 8 weeks following E12.5 liver transfer show typical LC (CD207, green) and DETC (TCRγδ, red) density and morphology when examined by confocal microscopy. Scalebar = 10 μm.
Figure 4
Figure 4. LC expression of CYP1B1 is necessary for optimal DMBA-induced DNA damage
(a) Ears of NLC and DTA controls as well as DTA repopulated with either w.t. or CYP1B1−/− LC were treated with 35 nmoles DMBA. 24 hours post-treatment epidermal sheets were prepared for γH2AX+ cell enumeration. Each dot represents one mouse. *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001. (b) Representative confocal images from (a) show LC (CD207, green) and nuclei containing DNA damage (γH2AX, red). Scalebar = 10μm.
Figure 5
Figure 5. LC expression of CYP1B1 is required for optimal cutaneous chemical carcinogenesis
(a) Chemical carcinogenesis was initiated in NLC and DTA controls as well as DTA repopulated with either w.t. or CYP1B1−/− LC using 400 nmoles DMBA followed by twice weekly TPA promotion (20 nmoles); ***P < 0.001 DTA+CYP1B1−/− LC vs DTA+w.t. LC and vs NLC; and P < 0.001 for DTA vs DTA+w.t. LC and vs NLC. N = 5–15 mice/group for panels a–c. (b) Representative images from (a) taken 12 weeks following initiation. (c) Percentage of tumor-free mice over time depicted in Kaplan-Meier plot. P < 0.05 for DTA vs DTA+CYP1B1−/− LC, P ≤ 0.01 for DTA+CYP1B1−/− LC vs DTA+w.t. LC and vs NLC, P < 0.0001 for DTA vs NLC and vs DTA+w.t.LC. (d,e) 14 weeks post initiation, DTA repopulated with CYP1B1−/− LC have more total tumors (d) and carcinomas (e) than LC-deficient DTA; n=7 DTA, 5 DTA+CYP1B1−/−LC. (f) Epidermal hyperplasia, as measured by minimal epidermal thickness, in response to repeated TPA application (4 weeks) is greater in the presence of LC; n=5 DTA, 5 NLC. (g) Ear thickness 48 hours after a single TPA application is greater in the presence of LC; n=6 DTA, 8 NLC. Baseline ear thickness (g) and minimal epidermal thickness (f) are not different in DTA vs NLC. *P <0.05, **P ≤ 0.01, ***P ≤ 0.001. Each dot represents one mouse in panels d–g.
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