Purpose: Basal cell carcinomas (BCCs) are tumors ignored by immune surveillance. Activated Hedgehog (Hh) signaling within primary cilia is a key driver in the pathogenesis of BCCs. We examined immune alterations during treatment with systemic Hh inhibitors.

Experimental Design: We investigated biopsies from patients with BCC before (23 patients) and after 4 weeks of treatment (5 patients) with Hh signaling inhibitor. Ber-Ep4, BCL-2, Ki-67, CD4, CD8, MHC class I, HLA-DR-class II, and SOX9 were analyzed by immunohistochemistry. Primary cilia were analyzed by double immunofluorescence of acetylated tubulin and SOX9. Differential gene expression for 84 cytokines and chemokines was analyzed in 3 patients.

Results: After 4 weeks of treatment, we found reduction of Ki-67, SOX9, Ber-EP4, and BCL-2 expression in tumors associated with morphologic signs of squamous differentiation. In addition, the number of cilia-positive BCC cells was significantly decreased. An upregulation of MHC I expression on the cell membranes of residual tumor cells and an influx of CD4+, HLA-DR-class II+, and CD8+ cells with invasion into the tumor cell nests were found. Finally, qPCR arrays showed the differential expression of genes involved in modulating immune responses.

Conclusions: We show that Hh pathway inhibitor–induced tumor regression is accompanied by a dynamic change of the microenvironment with a disruption of immune privilege involving an influx of cytotoxic T cells, activation of the adaptive immune functions, and a profound alteration of the local chemokine/cytokine network. Clin Cancer Res; 21(6); 1289–97. ©2015 AACR.

Translational Relevance

Basal cell carcinoma (BCC) is the most common malignancy in humans. Despite the presence of cancer testis antigens, BCCs are tumors that escape from adaptive immune surveillance. Because Hedgehog (Hh) pathway inhibitors are used in the clinic to treat patients with advanced or metastatic BCC, we investigated immune alterations during this systemic therapy. In this report, we show that Hh pathway inhibitor treatment reduces tumor burden and is accompanied by a recruitment of cytotoxic T cells into the tumor and reduction in the frequency of ciliated cells, which appear to be required for Hh inhibitor efficacy. We propose that these immune responses are crucial for long-term tumor control and hypothesize that a combination of Hh inhibitors with immune modifiers might be therapeutically beneficial.

Advanced basal cell carcinomas (BCCs) are a small subset of BCCs that cause significant morbidity and remain a therapeutic challenge due to their local invasiveness and proximity to vital structures (1). Despite the presence of cancer testis and other tumor antigens, BCCs escape from immune surveillance by the downregulation of HLA class I expression (2). Recently, targeted therapy based on knowledge of BCC pathogenesis has become available either commercially or in the context of human clinical trials. These orally available drugs, such as vismodegib and sonidegib, inhibit the Hedgehog (Hh) signaling pathway, and have improved the therapeutic repertoire (3–6).

The Hh pathway plays a crucial role in patterning and organogenesis during early development, and is largely inactive in the adult, except for its function in tissue repair and maintenance (7). The central components of the Hh pathway consist of three secreted ligands (Sonic Hh, Indian Hh, and Desert Hh), a negative regulatory receptor [Patched (PTCH)], a positive regulatory receptor [Smoothened (SMO), and glioma-associated oncogene (GLI) transcription factors (GLI1, GLI2, and GLI3; refs. 7, 8)]. Primary cilia are involved in a number of signaling cascades, including the Hh pathway (9). The primary cilium is a microtubule-based organelle that protrudes from the plasma membrane and acts as a sensor for extracellular signals. Hh signaling requires primary cilia. PTCH is located at the primary cilium in the absence of Hh; upon Hh binding, PTCH moves out of the cilium and SMO moves in and activates the GLI transcription factors (10, 11). In addition, a recent study showed that ciliary ablation strongly inhibited activated SMO-induced BCC-like tumors (12).

Vismodegib and sonidegib inhibit SMO and achieve significant tumor regressions including complete response in locally advanced and metastatic BCCs (3–6). Hh pathway activation was linked to BCC after the initial discovery of germline loss-of-function mutations in PTCH in patients with nevoid BCC syndromes (13). Most BCCs have mutations in the Hh signaling pathway that inactivate PTCH1 (loss-of-function mutation; refs. 8, 13) or, less commonly, constitutively activate SMO (gain-of-function mutation; ref. 14). These mutations cause active Hh pathway signaling, which in BCCs support proliferation of the neoplasia (3).

In this study, we demonstrate that inhibition of the Hh pathway results in upregulated MHC class I expression on BCCs and attracts MHC class II+, CD4+, and CD8+ T cells into the tumor cell nests. The in situ cytokine and chemokine networks are altered to an immune-supportive network, and cilia on BCCs were decreased during the treatment of Hh pathway inhibitors. This suggests that Hh pathway inhibitors promote adaptive immune reactions in BCCs.

Patients and treatments

We investigated biopsies of 23 patients with BCC treated with vismodegib (n = 22) or sonidegib (n = 1). Biopsies were collected after informed consent (EK number 647) was given. Patients were treated with vismodegib (150 mg daily) in the STEVIE study (a single-arm, open-label, phase II, multicenter study to assess the safety of vismodegib in patients with locally advanced or metastatic BCC) or with sonedigib (200 or 800 mg daily) in the BOLT study (a phase II study of efficacy and safety in patients with locally advanced or metastatic BCC; ref. 15).

The overall response rate in those patients with measurable disease was according to RECIST Version 1.1 (RECIST, v1.1). Evaluation of target lesions was performed as below. Complete response: disappearance of all target lesions. Any pathologic lymph nodes (whether target or nontarget) must have reduction in short axis to <10 mm. Partial response: at least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters.

Punch biopsies (3–6 mm) were taken before (23 samples) treatment initiation and after 4 weeks (5 samples, detailed information on the patients: Table 1) of treatment. Punch biopsies were outside the initial biopsy areas.

Table 1.

Clinical presentation and histology of the 5 patients with paired biopsies

PatientClinical presentationHistology before treatmentHistology after 4 weeksTumor size (after 4 weeks and 8 weeks)Best response after weeks of vismodegib treatment
Locally advanced, facial, ulcerated, 150 × 80 mm measuring BCC Solid BCC Regression of tumor with inflammatory infiltrate 120 × 80 mm, 120 × 80 mm CR after 16 weeks 
Locally advanced, thoracal, ulcerated, 80 × 60 mm measuring BCC Solid BCC Regression of tumor with inflammatory infiltrate 80 × 60 mm, 50 × 40 mm PR after 12 weeks 
Locally advanced, facial, ulcerated 50 × 60 mm measuring BCC BCC with basosquamous differentiation Epithelial tumor rests infiltrated by inflammatory cells 40 × 40 mm, 40 × 35 mm PR after 16 weeks 
Multiple locally advanced, recurrent BCC, 10 × 12 mm with lower leg Superficial BCC Superficial tumor rests and inflammatory infiltrate 10 × 12 mm, 10 × 10 mm PR after 12 weeks 
Multiple BCC, 45 × 15 mm with upper arm Superficial BCC Regression of BCC with focal basosquamous differentiation, inflammatory infiltrate, and cystic formation 45 × 15 mm, 45 × 10 mm MR after 12 weeks 
PatientClinical presentationHistology before treatmentHistology after 4 weeksTumor size (after 4 weeks and 8 weeks)Best response after weeks of vismodegib treatment
Locally advanced, facial, ulcerated, 150 × 80 mm measuring BCC Solid BCC Regression of tumor with inflammatory infiltrate 120 × 80 mm, 120 × 80 mm CR after 16 weeks 
Locally advanced, thoracal, ulcerated, 80 × 60 mm measuring BCC Solid BCC Regression of tumor with inflammatory infiltrate 80 × 60 mm, 50 × 40 mm PR after 12 weeks 
Locally advanced, facial, ulcerated 50 × 60 mm measuring BCC BCC with basosquamous differentiation Epithelial tumor rests infiltrated by inflammatory cells 40 × 40 mm, 40 × 35 mm PR after 16 weeks 
Multiple locally advanced, recurrent BCC, 10 × 12 mm with lower leg Superficial BCC Superficial tumor rests and inflammatory infiltrate 10 × 12 mm, 10 × 10 mm PR after 12 weeks 
Multiple BCC, 45 × 15 mm with upper arm Superficial BCC Regression of BCC with focal basosquamous differentiation, inflammatory infiltrate, and cystic formation 45 × 15 mm, 45 × 10 mm MR after 12 weeks 

Abbreviations: CR, complete remission according protocol; MR, minor remission (not qualifying for PR, but clinical improvement); PR, partial remission according to protocol.

Immunohistochemistry

All tissues used for immunohistochemistry were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were deparaffinized in xylene and rehydrated. Epitope retrieval was performed in antibody-specific buffers. The following antibodies were used: Ber-Ep4, Bcl-2, Ki-67, CD8 (DAKO), CD4, HLA-DR-class II (Novocastra), MHC class I (RDI Research Diagnostics), CD68 (DAKO), Foxp3 (Abcam), and Sox9 (Millipore). Staining was performed using kits supplied by Ventana or Dako REAL Detection System (kit 5005). Antigen-specific antibodies were applied and visualized with either the iVIEW DAB detection Kit (Ventana) or the ChemMate detection Kit (Dako). Slides were counterstained with hematoxylin and eosin (H&E). The figures show representative paired pre- and posttreatment samples.

Immunofluorescence

Sections were deparaffinized with xylene and rehydrated with 70% EtOH. Antigen retrieval was performed at 110°C in a pressure cooker for 5 minutes in citrate buffer. Blocking was performed for 30 minutes at room temperature in blocking solution (10% goat serum, 0.3% Tween). Incubation was carried out with the following primary antibodies: acetylated Tubulin (T6793; Sigma; 1:1,000 dilution) and Sox9 (sc-20095; Santa Cruz Biotechnology; 1:50 dilution). Incubation was carried out with the following secondary antibodies: Alexa-488 goat anti-mouse (Life Technologies; 1:1,000 dilution) and Alex-568 goat anti-rabbit (Life Technologies; 1:100 dilution) followed by DAPI (500 ng/mL). The results showed representative paired pre- and posttreatment samples.

RT-PCR

Total RNA was extracted from a punch of frozen tumor material using TRIzol (Invitrogen) according to manufacturer's instructions. cDNA was made from the RT2 HT First Strand Kit (330441; Qiagen) according to manufacturer's instructions. Genes were evaluated with the Human cytokine & chemokine PCR array (PAHS-150ZA; Qiagen) with the Viia7 system from Applied Biosystems. Fold change and P values were calculated by RT2 Profiler PCR Array Analysis (Qiagen). Genes were normalized to the three housekeeping genes on the array.

Image analysis and quantification

Images of the stained paraffin sections were acquired with Scanscope Image (Image Scope; Aperio Technologies). Quantification of CD4, CD8, HLA-DR-class II, CD68, and Foxp3+ cells in intratumoral and peritumoral regions was done by counting cells in high-power fields (HPF) of ×40 magnification. For each section, 4 × 40 HPF representative areas per sample were counted. Immunoreactivity of Ki67 (nuclear), Sox9 (nuclear), BerEP (cytoplasm), Bcl2 (cytoplasm), and MHC class I expression (membrane of tumor cells) on tumor cells was quantified by investigation of 4 × 40 HPF representative areas per sample and evaluated as grade 1 (<10% of cells), grade 2 (10%–30% of cells), and grade 3 (>30% of cells). The number of cilia-positive cells in each section was determined by counting cells in HPFs of ×100 magnification. The nucleated cells are counted in all experiments. A point on the immunological figures represents each single counted field.

Statistical analysis

Data of immunological studies and RT-PCR are presented as the mean ± SD. RT-PCR data include data of three independent experiments. P values were calculated with a parametric Student t test. *, P < 0.05.

Reduction of Ki67, Sox9, Ber-EP4, Bcl-2, and cilium expressions on BCC after the treatment with Hh pathway inhibitors

In all 23 pretreatment biopsies, diagnosis of BCC was histologically confirmed in HE-stained sections by a board-certified dermatopathologist (R. Dummer). Twenty-three patients (10 male, 13 female; 46–90 years; mean, 70 years) were all partial responders of Hh pathway inhibitor treatment. Further investigation was focused on 5 patients (Table 1), with paired biopsies taken before and after 4 weeks of treatment with Hh pathway inhibitors, who consented to the biopsy for clinical research. All patients suffered from locally advanced BCC without Gorlin syndrome. Clinical presentation and histologic diagnosis of these patients are summarized in Table 1. In all cases, comparison of HE-stained sections from before and after 4 weeks of Hh pathway inhibitor treatment showed a tumor regression with a reduction of the tumor nest (Fig. 1A). The morphology was altered with a more eosinophilic staining pattern and signs of cornification suggesting a transdifferentiation into a squamous phenotype. In one case, we observed the formation of cytic structures filled with keratin (Fig. 1A).

Figure 1.

Histology and immunohistochemical stainings of Ki67, Sox9, BerEP, and bcl2. A, HE staining and Ki67, Sox9, BerEP, and bcl2 by immunohistochemical staining of a representative pair of biopsies with different HPFs before and after 4 weeks of treatment. Scale bar, 100 μm. B Ki67, BerEP, Bcl2, and Sox9 expression on tumor cells was quantified by investigation of 4 × 40 HPF representative of different patients and different HPFs per sample and evaluated as grade 1 (<10% of cells or none staining for Sox9), grade 2 (10%–30% of cells or weak staining for Sox9), and grade 3 (>30% of cells or strong staining for Sox9).

Figure 1.

Histology and immunohistochemical stainings of Ki67, Sox9, BerEP, and bcl2. A, HE staining and Ki67, Sox9, BerEP, and bcl2 by immunohistochemical staining of a representative pair of biopsies with different HPFs before and after 4 weeks of treatment. Scale bar, 100 μm. B Ki67, BerEP, Bcl2, and Sox9 expression on tumor cells was quantified by investigation of 4 × 40 HPF representative of different patients and different HPFs per sample and evaluated as grade 1 (<10% of cells or none staining for Sox9), grade 2 (10%–30% of cells or weak staining for Sox9), and grade 3 (>30% of cells or strong staining for Sox9).

Close modal

We examined the expression of tumor- or BCC-associated proteins by immunohistochemical staining. In all cases, we found dramatic reduction of the expression of the proliferation marker Ki-67, the stemness marker Sox9, the hair follicle–specific antigen Ber-EP4, and the antiapoptotic molecule Bcl-2 on the tumor cells after 4 weeks of treatment with Hh pathway inhibitors (Fig. 1A and B). This was in one case associated with cyst formation.

The primary cilium has emerged as an important organelle that is required for Hh signaling (9). However, it is still unknown how cilia change on human BCCs during Hh pathway inhibitor treatment. Therefore, we next examined the expression of cilia that are identified by acetylated tubulin. In all cases investigated, we found ciliated BCC cells by immunofluorescence. Intriguingly, the number of cilia-positive cells on BCC is significantly decreased after 4 weeks of treatment compared with that before Hh pathway inhibitor treatment (Fig. 2). We confirmed that there was no nonspecific staining for cilia (Supplementary Fig. S1A).

Figure 2.

Expression of cilia and SOX9. A, cilia and Sox9 by immunohistochemical staining of a representative biopsy pair before and after 4 weeks treatment. B, 3 × 100 HPF representative areas per sample were counted in BCC biopsies from patients before and after 4 weeks of treatment with Hh inhibitors (n = 4). *, P < 0.05.

Figure 2.

Expression of cilia and SOX9. A, cilia and Sox9 by immunohistochemical staining of a representative biopsy pair before and after 4 weeks treatment. B, 3 × 100 HPF representative areas per sample were counted in BCC biopsies from patients before and after 4 weeks of treatment with Hh inhibitors (n = 4). *, P < 0.05.

Close modal

Promotion of adaptive immune cell infiltration and upregulation of MHC class I in BCCs following Hh inhibitor treatment

We next analyzed the effect of Hh pathway inhibitor treatment on immune reactions in BCCs. In pretreatment biopsies, CD68+, CD4+, CD8+, and FoxP3+ cells were rare and solely located in the stroma. Immunohistological staining revealed an increase and invasion of CD8+ T cells into tumor cell nests (Fig. 3A) with an upregulation of MHC class I expression on tumor cell membranes (Fig 3B; Supplementary Fig. S2). Interestingly, at week 4, an increase of peri- and intratumoral CD4+ T cells was detected in the tumor area (Fig. 3C). In addition, the number of HLA-DR-class II+ mononuclear cells was significantly increased in the intratumoral and peritumoral areas after 4 weeks of treatment (Fig. 3D).

Figure 3.

CD4 (C), CD8 (A), HLA-DR-class II (D), and MHC class I expression. (B) MHC class I expression on tumor cells was quantified by investigation of 4 × 40 HPF of a representative biopsy pair and evaluated as grade 1 (<10% of cells), grade 2 (10%–30% of cells), and grade 3 (>30% of cells). Quantification of CD4, CD8, and HLA-DR-class II+ cells in intratumoral and peritumoral regions was done by counting cells in HPFs of ×40 magnification. For each section, 4 × 40 HPF representative areas per sample were counted from patients before and after 4 weeks of treatment with Hh inhibitors. *, P < 0.05.

Figure 3.

CD4 (C), CD8 (A), HLA-DR-class II (D), and MHC class I expression. (B) MHC class I expression on tumor cells was quantified by investigation of 4 × 40 HPF of a representative biopsy pair and evaluated as grade 1 (<10% of cells), grade 2 (10%–30% of cells), and grade 3 (>30% of cells). Quantification of CD4, CD8, and HLA-DR-class II+ cells in intratumoral and peritumoral regions was done by counting cells in HPFs of ×40 magnification. For each section, 4 × 40 HPF representative areas per sample were counted from patients before and after 4 weeks of treatment with Hh inhibitors. *, P < 0.05.

Close modal

Moreover, the number of CD68+ macrophages was significantly increased in the intratumoral and peritumoral areas after 4 weeks of treatment (Fig. 4A). BCC has been shown to be associated with regulatory T cells (Treg) that could contribute to an immunosuppressive activity against tumor-specific T-cell responses (16). Therefore, we next analyzed the number of Foxp3+ Tregs. The number of Tregs was significantly increased in the intratumoral and peritumoral areas after 4 weeks of treatment (Fig. 4B). Interestingly, the ratio CD8+/Foxp3+ was increased only in the intratumoral but not in peritumoral areas (Fig. 4C). We confirmed that there was no nonspecific staining (Supplementary Fig. S1B).

Figure 4.

Immunohistochemical stainings of CD68 and Foxp3.A and B, for CD68 and Foxp3 staining, 4 × 40 HPFs of a representative biopsy pair are shown, and 5 pairs were counted in BCC biopsies from patients before and after 4 weeks of treatment with Hh inhibitors (n = 5). *, P < 0.05. C, the ratio CD8+/Foxp3+ was increased only in the intratumoral but not in peritumoral areas.

Figure 4.

Immunohistochemical stainings of CD68 and Foxp3.A and B, for CD68 and Foxp3 staining, 4 × 40 HPFs of a representative biopsy pair are shown, and 5 pairs were counted in BCC biopsies from patients before and after 4 weeks of treatment with Hh inhibitors (n = 5). *, P < 0.05. C, the ratio CD8+/Foxp3+ was increased only in the intratumoral but not in peritumoral areas.

Close modal

Change of the cytokine and chemokine milieu after Hh pathway treatment

It is likely that there is an immunological change after the treatment of Hh pathway inhibitors. To address this issue, we performed qRT-PCR using an array that included 84 cytokines and chemokines. The results showed 6 genes to be differentially expressed after Hh pathway inhibitor treatment. Of these 6 genes, 5 were upregulated by at least 2-fold after the treatment and 1 was downregulated at least 2-fold after treatment (Fig. 5). There was a significant increase of the expression levels of the chemokines and cytokines: chemokine (C-C motif) ligand (CCL)18, CCL21, chemokine (C-X-C motif) ligand (CXCL) 9, vascular endothelial growth factor A (VEGFA), and TNF ligand superfamily member 11 (TNFSF11, receptor activator of nuclear factor kappa-B ligand, RANKL). Furthermore, the qPCR results showed a consistent decrease during treatment of the expression levels of TNF receptor superfamily member 11B (TNFRSF11B, osteoprotegerin; Fig. 5). The expression level of IFNγ increased 1.63-fold, but this was not significant.

Figure 5.

Changes in cytokine profile PCR array of paired biopsies (n = 3) before and after 4 weeks of treatment with Hh inhibitors showed an increase of the expression levels of different chemokines and cytokines: CCL18, CCL21, CXCL 9, VEGFA, and TNFSF11 (RANKL), and consistent decrease during treatment of the expression levels of TNFSF11 (RANKL) receptor TNFRSF11B. *, P < 0.05.

Figure 5.

Changes in cytokine profile PCR array of paired biopsies (n = 3) before and after 4 weeks of treatment with Hh inhibitors showed an increase of the expression levels of different chemokines and cytokines: CCL18, CCL21, CXCL 9, VEGFA, and TNFSF11 (RANKL), and consistent decrease during treatment of the expression levels of TNFSF11 (RANKL) receptor TNFRSF11B. *, P < 0.05.

Close modal

This study reports on the histologic alterations on the tumor cell population and the inflammatory tumor microenvironment of patients with BCCs as a result of Hh inhibitor treatment. These data provide evidence that the Hh pathway–induced tumor regression leads to a dramatic change in the microenvironment that is characterized with a disruption of immune privilege and the activation of the adaptive immune effector functions.

A previous study demonstrated that vismodegib showed a 30% and 60% response rate for metastatic and locally advanced BCC (5). However, most responses were partial responses. In addition, significant adverse events with negative impact on the quality of life, including muscle spasms, alopecia, dysgeusia, weight loss, and fatigue, were occurred in more than 30% of patients. Therefore, improvement of the therapy using Hh pathway inhibitors is required. In this study, we demonstrated that Hh pathway inhibitors promoted adaptive immunity via upregulation of MHC class I and infiltrating immune cells into the tumor sites. A combination therapy with immune modifiers has a possibility to improve the efficiency by shortening the treatment-related drug exposure and achieving a complete remission rate.

We demonstrated substantial alterations in the immune microenvironment with an intra- and peritumoral increase of CD4+ and cytotoxic CD8+ T cells and an upregulation of MHC class I during tumor regression under treatment with Hh pathway inhibitors. In addition, reduction of primary cilia was observed after Hh pathway inhibitor treatment. Primary cilia have been implicated in Hh pathway signaling, and ablation of cilia in SMO-activated cells inhibits tumor growth (17). Before treatment, all BCC cells are ciliated, suggesting that they are responsive to Hh signaling. By inhibiting Hh signaling, the BCC cells lose their cilia and subsequently stop proliferating, as seen in the reduction of Ki-67 staining.

T-cell activation requires both T-cell receptor (TCR) and costimulatory molecule ligation by professional antigen-presenting cells (APC), and the outcome of the stimulatory signal is influenced by the microenvironment of the T cell and the APC. The Hh signaling pathway reduced the strength of the TCR signal in mature peripheral T cells (18, 19). In addition, the repression of the Hh signaling pathway in T cells increased T-cell activation (20). This suggests that the Hh pathway inhibitor has direct effects on peripheral T cell and activates adaptive immune responses. The precise mechanism of the Hh inhibitor on immune modulation requires further investigation.

RT-PCR results showed that the expression levels of different chemo- and cytokines changed during treatment (Fig. 5). Although these are limited in number, our data suggest that these changes in the immune environment may be crucial for tumor control and highlight the poorly understood interface between targeted therapy and immune responses. BCCs frequently express multiple cancer testis antigens, but are also associated with relative absence of MHC class I molecules from tumor cells, and it has been hypothesized that BCCs produce immunosuppressive factors such as IL10 (2, 21) and therefore are resistant to adaptive immune response. This might explain the absence of infiltrating CD8+ cells in BCC. We found that the chemokines CCL18, CCL21, and CXCL9 were upregulated during treatment with Hh pathway inhibitors. These chemokines are produced and secreted by innate immune cells, such as macrophages, and exert their effect mainly on the adaptive immune system. It is known that epithelial cells can produce CCL18, CCL21, and CXCL9 (22–24). These chemokines are thought to have a critical role in tumor suppression. Interestingly, high CCL18 or CCL21 expression was reported as a favorable prognostic factor in patients with colorectal cancer (25, 26). In addition, we found an upregulation of VEGFA during treatment, which may support granulation reactions with angiogenesis in regressive BCC lesions.

Although the expression level of IFNγ increased 1.63-fold, and was not significant in this study, our current findings provide an indirect indication for an IFNγ-primed microenvironment favoring immune response. It is known that IFNγ upregulates MHC class I antigen presentation by inducing gene expression signatures that are related to MHC class I antigen processing and presentation including activation of JAK/STAT1 signal transduction pathway (27). Furthermore, changes in cytokine profiles with upregulation of CXCL9 [also known as Monokine induced by gamma interferon (MIG)], which has chemotactic activity on T cells induced by IFNγ, support this hypothesis. A potential cross-talk between IFNγ and the Hh pathway was recently described by Laner-Plamberger and colleagues (28). They demonstrated that suppressor of cytokine signaling 1 (SOCS1) is a direct target of Hh/GLI signaling in human keratinocytes and medulloblastoma cells and a potent inhibitor of IFNγ–STAT1 signaling, which can induce cell cycle arrest, apoptosis, and antitumor immunity. It was shown that the transcription factors GLI1 and GLI2 activated the SOCS1 promoter, and STAT1 phosphorylation was reduced in cells with active Hh/GLI signaling (28). mRNA levels including IFNγ depend on when the samples are collected (29, 30). A previous report showed that IFNγ mRNA expression in the skin-infiltrated IFNγ-producing T cells increased at the peak of 12 hours and diminished after 24 hours in skin allergic inflammation (30). In addition to the limited number of samples in this work, the biopsy time point may be too late to directly document the IFNγ peak.

In vitro studies on bone tissue showed that Hh signaling indirectly induced osteoclast differentiation by upregulating osteoblast expression of parathyroid hormone–related protein (PTHrP), which promoted RANKL expression (31). Mouse models on human bone tissue emphasized these results. RANKL was induced by activation of the Hh pathway, and the expression of RANKL was inversely associated with that of HLA-G5, especially with Hh stimulation (32). Our analyses of cytokine profiles showed an upregulation of RANKL during treatment with Hh inhibitors. RANKL is also expressed by Th cells and is thought to be involved in dendritic cell maturation as well as the regulation of T-cell–dependent immune responses.

In conclusion, we demonstrated that Hh pathway inhibitor treatment–induced tumor regression was accompanied by a recruitment of cytotoxic T cells into the tumor and reduction in the frequency of ciliated cells, which appeared to be required for Hh inhibitor efficacy. Therefore, we propose that these immune responses are crucial for long-term tumor control and hypothesize that a combination of Hh inhibitors with immune modifiers might be therapeutically beneficial.

R. Dummer is a consultant/advisory board member for Roche and Novartis. No potential conflicts of interest were disclosed by the other authors.

Conception and design: A. Otsuka, P.F. Cheng, M. Nägeli, I.J. Frew, M.P. Levesque, R. Dummer

Development of methodology: M. Nägeli, M.P. Levesque, R. Dummer

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Otsuka, P.F. Cheng, M. Nägeli, H. Lehmann, L. Felderer, R. Dummer

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Otsuka, J. Dreier, P.F. Cheng, M. Nägeli, I.J. Frew, M.P. Levesque, R. Dummer

Writing, review, and/or revision of the manuscript: A. Otsuka, J. Dreier, P.F. Cheng, M. Nägeli, H. Lehmann, L. Felderer, I.J. Frew, S. Matsushita, M.P. Levesque, R. Dummer

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Nägeli, L. Felderer, M.P. Levesque, R. Dummer

Study supervision: M. Nägeli, L. Felderer, M.P. Levesque, R. Dummer

The authors thank Ines Kleiber-Schaaf for technical assistance.

This work was supported in part by the Zürich University Research Priority Program (URPP) Translational Cancer Research (U-402), the Society for Skin Cancer Research (to M.P. Levesque; F-85803-03-01), and the Schwyzer Foundation (P13.28).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Dreier
J
,
Cheng
PF
,
Bogdan Alleman
I
,
Gugger
A
,
Hafner
J
,
Tschopp
A
, et al
Basal cell carcinomas in a tertiary referral centre: a systematic analysis
.
Br J Dermatol
2014
;
171
:
1066
72
.
2.
Walter
A
,
Barysch
MJ
,
Behnke
S
,
Dziunycz
P
,
Schmid
B
,
Ritter
E
, et al
Cancer-testis antigens and immunosurveillance in human cutaneous squamous cell and basal cell carcinomas
.
Clin Cancer Res
2010
;
16
:
3562
70
.
3.
Von Hoff
DD
,
LoRusso
PM
,
Rudin
CM
,
Reddy
JC
,
Yauch
RL
,
Tibes
R
, et al
Inhibition of the hedgehog pathway in advanced basal-cell carcinoma
.
N Engl J Med
2009
;
361
:
1164
72
.
4.
Rodon
J
,
Tawbi
HA
,
Thomas
AL
,
Stoller
RG
,
Turtschi
CP
,
Baselga
J
, et al
A phase I, multicenter, open-label, first-in-human, dose-escalation study of the oral smoothened inhibitor Sonidegib (LDE225) in patients with advanced solid tumors
.
Clin Cancer Res
2014
;
20
:
1900
9
.
5.
Sekulic
A
,
Migden
MR
,
Oro
AE
,
Dirix
L
,
Lewis
KD
,
Hainsworth
JD
, et al
Efficacy and safety of vismodegib in advanced basal-cell carcinoma
.
N Engl J Med
2012
;
366
:
2171
9
.
6.
Tang
JY
,
Mackay-Wiggan
JM
,
Aszterbaum
M
,
Yauch
RL
,
Lindgren
J
,
Chang
K
, et al
Inhibiting the hedgehog pathway in patients with the basal-cell nevus syndrome
.
N Engl J Med
2012
;
366
:
2180
8
.
7.
Varjosalo
M
,
Taipale
J
. 
Hedgehog: functions and mechanisms
.
Genes Dev
2008
;
22
:
2454
72
.
8.
Johnson
RL
,
Rothman
AL
,
Xie
J
,
Goodrich
LV
,
Bare
JW
,
Bonifas
JM
, et al
Human homolog of patched, a candidate gene for the basal cell nevus syndrome
.
Science
1996
;
272
:
1668
71
.
9.
Wong
SY
,
Reiter
JF
. 
The primary cilium at the crossroads of mammalian hedgehog signaling
.
Curr Top Dev Biol
2008
;
85
:
225
60
.
10.
Corbit
KC
,
Aanstad
P
,
Singla
V
,
Norman
AR
,
Stainier
DY
,
Reiter
JF
. 
Vertebrate smoothened functions at the primary cilium
.
Nature
2005
;
437
:
1018
21
.
11.
Rohatgi
R
,
Milenkovic
L
,
Scott
MP
. 
Patched1 regulates hedgehog signaling at the primary cilium
.
Science
2007
;
317
:
372
6
.
12.
Liao
X
,
Siu
MK
,
Au
CW
,
Chan
QK
,
Chan
HY
,
Wong
ES
, et al
Aberrant activation of hedgehog signaling pathway contributes to endometrial carcinogenesis through beta-catenin
.
Mod Pathol
2009
;
22
:
839
47
.
13.
Hahn
H
,
Wicking
C
,
Zaphiropoulous
PG
,
Gailani
MR
,
Shanley
S
,
Chidambaram
A
, et al
Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome
.
Cell
1996
;
85
:
841
51
.
14.
Xie
J
,
Murone
M
,
Luoh
SM
,
Ryan
A
,
Gu
Q
,
Zhang
C
, et al
Activating smoothened mutations in sporadic basal-cell carcinoma
.
Nature
1998
;
391
:
90
2
.
15.
Grob
JJ
,
Kunstfeld
R
,
Dreno
B
,
Jouary
T
,
Mortier
L
,
Basset-Seguin
N.
Vismodegib, a Hedgehog pathway inhibitor (HPI), in advanced basal cell carcinoma (aBCC): STEVIE study interim analysis in 300 patients
.
J Clin Oncol
2013
;
31
:
9036
.
16.
Kaporis
HG
,
Guttman-Yassky
E
,
Lowes
MA
,
Haider
AS
,
Fuentes-Duculan
J
,
Darabi
K
, et al
Human basal cell carcinoma is associated with Foxp3+ T cells in a Th2 dominant microenvironment
.
J Invest Dermatol
2007
;
127
:
2391
8
.
17.
Wong
SY
,
Seol
AD
,
So
PL
,
Ermilov
AN
,
Bichakjian
CK
,
Epstein
EH
 Jr
, et al
Primary cilia can both mediate and suppress Hedgehog pathway-dependent tumorigenesis
.
Nat Med
2009
;
15
:
1055
61
.
18.
Rowbotham
NJ
,
Hager-Theodorides
AL
,
Cebecauer
M
,
Shah
DK
,
Drakopoulou
E
,
Dyson
J
, et al
Activation of the Hedgehog signaling pathway in T-lineage cells inhibits TCR repertoire selection in the thymus and peripheral T-cell activation
.
Blood
2007
;
109
:
3757
66
.
19.
Rowbotham
NJ
,
Hager-Theodorides
AL
,
Furmanski
AL
,
Crompton
T
. 
A novel role for Hedgehog in T-cell receptor signaling: implications for development and immunity
.
Cell Cycle
2007
;
6
:
2138
42
.
20.
Rowbotham
NJ
,
Furmanski
AL
,
Hager-Theodorides
AL
,
Ross
SE
,
Drakopoulou
E
,
Koufaris
C
, et al
Repression of hedgehog signal transduction in T-lineage cells increases TCR-induced activation and proliferation
.
Cell Cycle
2008
;
7
:
904
8
.
21.
Nestle
FO
,
Burg
G
,
Fah
J
,
Wrone-Smith
T
,
Nickoloff
BJ
. 
Human sunlight-induced basal-cell-carcinoma-associated dendritic cells are deficient in T cell co-stimulatory molecules and are impaired as antigen-presenting cells
.
Am J Pathol
1997
;
150
:
641
51
.
22.
Ichimura
M
,
Hiratsuka
K
,
Ogura
N
,
Utsunomiya
T
,
Sakamaki
H
,
Kondoh
T
, et al
Expression profile of chemokines and chemokine receptors in epithelial cell layers of oral lichen planus
.
J Oral Pathol Med
2006
;
35
:
167
74
.
23.
Lkhagvasuren
E
,
Sakata
M
,
Ohigashi
I
,
Takahama
Y
. 
Lymphotoxin beta receptor regulates the development of CCL21-expressing subset of postnatal medullary thymic epithelial cells
.
J Immunol
2013
;
190
:
5110
7
.
24.
Huang
W
,
Hu
K
,
Luo
S
,
Zhang
M
,
Li
C
,
Jin
W
, et al
Herpes simplex virus type 2 infection of human epithelial cells induces CXCL9 expression and CD4+ T cell migration via activation of p38-CCAAT/enhancer-binding protein-beta pathway
.
J Immunol
2012
;
188
:
6247
57
.
25.
Yuan
R
,
Chen
Y
,
He
X
,
Wu
X
,
Ke
J
,
Zou
Y
, et al
CCL18 as an independent favorable prognostic biomarker in patients with colorectal cancer
.
J Surg Res
2013
;
183
:
163
9
.
26.
Zou
Y
,
Chen
Y
,
Wu
X
,
Yuan
R
,
Cai
Z
,
He
X
, et al
CCL21 as an independent favorable prognostic factor for stage III/IV colorectal cancer
.
Oncol Rep
2013
;
30
:
659
66
.
27.
Zhou
F
. 
Molecular mechanisms of IFN-γ to up-regulate MHC class I antigen processing and presentation
.
Int Rev Immuno
2009
;
28
:
239
60
.
28.
Laner-Plamberger
S
,
Wolff
F
,
Kaser-Eichberger
A
,
Swierczynski
S
,
Hauser-Kronberger
C
,
Frischauf
AM
, et al
Hedgehog/GLI signaling activates suppressor of cytokine signaling 1 (SOCS1) in epidermal and neural tumor cells
.
PLoS One
2013
;
8
:
e75317
.
29.
Otsuka
A
,
Nakajima
S
,
Kubo
M
,
Egawa
G
,
Honda
T
,
Kitoh
A
, et al
Basophils are required for the induction of Th2 immunity to haptens and peptide antigens
.
Nat Commun
2013
;
4
:
1739
.
30.
Kitagaki
H
,
Ono
N
,
Hayakawa
K
,
Kitazawa
T
,
Watanabe
K
,
Shiohara
T
. 
Repeated elicitation of contact hypersensitivity induces a shift in cutaneous cytokine milieu from a T helper cell type 1 to a T helper cell type 2 profile
.
J Immunol
1997
;
159
:
2484
91
.
31.
Mak
KK
,
Bi
Y
,
Wan
C
,
Chuang
PT
,
Clemens
T
,
Young
M
, et al
Hedgehog signaling in mature osteoblasts regulates bone formation and resorption by controlling PTHrP and RANKL expression
.
Dev Cell
2008
;
14
:
674
88
.
32.
Deschaseaux
F
,
Gaillard
J
,
Langonné
A
,
Chauveau
C
,
Naji
A
,
Bouacida
A
, et al
Regulation and function of immunosuppressive molecule human leukocyte antigen G5 in human bone tissue
.
FASEB J
2013
;
27
:
2977
87
.