FcRgamma activation regulates inflammation-associated squamous carcinogenesis

Abstract

Chronically activated leukocytes recruited to premalignant tissues functionally contribute to cancer development; however, mechanisms underlying pro- versus anti-tumor programming of neoplastic tissues by immune cells remain obscure. Using the K14-HPV16 mouse model of squamous carcinogenesis, we report that B cells and humoral immunity foster cancer development by activating Fcgamma receptors (FcgammaRs) on resident and recruited myeloid cells. Stromal accumulation of autoantibodies in premalignant skin, through their interaction with activating FcgammaRs, regulate recruitment, composition, and bioeffector functions of leukocytes in neoplastic tissue, which in turn promote neoplastic progression and subsequent carcinoma development. These findings support a model in which B cells, humoral immunity, and activating FcgammaRs are required for establishing chronic inflammatory programs that promote de novo carcinogenesis.

Figure 1. B Cells Are Critical Regulators of Premalignant Progression in HPV16 Mice

(A) Percentage of CD45⁺ cells in skin single cell suspensions isolated from negative littermates (−LM), HPV16/JH^+/−, and HPV16/JH^−/− mice at 1, 4, and 6 months of age assessed by flow cytometry. (B and C) Mast cells (B, blue staining) and Gr1⁺ myeloid cells (C, brown staining) in skin of HPV16/JH^+/− and HPV16/JH^−/− mice at 1, 4, and 6 months of age assessed quantitatively after chloroacetate esterase histochemistry or Gr1 immunohistochemistry (IHC), respectively. (D) Flow cytometric analysis of immune cell lineages expressed as percentages of total CD45⁺ leukocyte infiltrates in ear tissue of negative littermates (−LM), HPV16, HPV16/JH^+/−, HPV16/JH^−/−, and HPV16/RAG1^−/− mice at 1, 4, and 6 months of age. (E) Dendritic (CD11c⁺) and macrophage (F4/80⁺) lineage cell composition of CD11b⁺Gr1⁺ (left) and CD11b⁺Gr1⁻ (right) myeloid populations evaluated by flow cytometry in skin of negative littermates (−LM), HPV16, and HPV16/JH^−/− mice at 4 months of age. (F) Angiogenic vasculature in skin tissue sections from negative littermates (−LM), HPV16/JH^+/−, and HPV16/JH^−/− mice at 1, 4, and 6 months of evaluation by CD31/PECAM-1 IHC revealing endothelial cells (brown staining). (G) Reduced VEGF-A and active MMP-9 protein levels in skin extracts from HPV16/JH^−/− versus HPV16/JH^+/− mice (4 and 6 months) as assessed by ELISA. (H) Keratinocyte proliferation in skin of negative littermates (−LM), HPV16/JH^+/−, and HPV16/JH^−/− mice at 1, 4, and 6 months of age, as evaluated by quantitation of bromodeoxyuridine (BrdU)-positive keratinocytes (red staining). (I) Percentage of ear skin in HPV16/JH^−/− and HPV16/JH^+/− mice developing hyperplastic lesions by 1 month of age (Hyp) or dysplasia by 4 and 6 months of age (Dys). (A–I) Results shown represent mean ± SEM (n = 5–8 mice) and asterisks (*) indicate statistically significant differences (p < 0.05, Mann-Whitney). Representative images of HPV16/JH^+/− and HPV16/JH^−/− mouse skin at 4 months of age are shown. Values represent average of five high-power fields of view per mouse and five mice per category. FOV, field of view; solid red line, epidermal-dermal interface; e, epidermis; d, dermis. Scale bars represent 50 μm. See also Figure S1.

Figure 2. CICs from HPV16 Mice Induce Acute Inflammation

(A) CICs titers in serum collected from negative littermate (−LM), HPV16/JH^+/+, and HPV16/JH^−/− mice assessed by ELISA with anti-mouse C3 Ig (left) and C1q (right). The specificity of C1q/IgG binding to ICs was assessed by disrupting C1q-CIC binding using a high-salt solution (C1q/IC/d; gray bars). (B) Antibody titers for laminin (Lm)-111, Lm-332, collagen (Col)-I, Col-II, and Col-IV in serum from littermate (−LM), HPV16, and HPV16/JH^−/− (4 months) were evaluated by indirect ELISA. Antigens recognized by biotinylated IgG_HPV16 were localized by immunofluorescence on skin sections from 4-month-old HPV16 mice (inset; IgGs: red staining). (C) Immunofluorescence (green staining) staining on syngeneic skin tissue sections reveals presence of intradermally injected purified IgGs isolated from negative littermate (IgG_wt) or HPV16 (IgG_HPV16) 24 hr after intradermal injections. (D) Number of infiltrating CD45⁺ leukocytes and Gr1⁺ cells in skin tissue sections 24 hr after intradermal injections of PBS, IgG_wt, or IgG_HPV16 into ears of syngeneic wild-type FVB/n mice. Values reflect averages from five high-power fields per mouse. (E) Normalized CIC titers of IgG purified from negative littermates (IgG_wt) or HPV16 (IgG_HPV16) serum assessed by ELISA with anti-mouse C3 Ig (left) and C1q (right). (B and C) Blue staining, DAPI; white line, epidermal-dermal interface; e, epidermis; d, dermis. Scale bars represent 50 μm. (B–D) Results shown are mean percentages ± SEM (n = 5–8 mice) and asterisks (*) indicate statistically significant differences (p < 0.05, Mann-Whitney). See also Figure S2.

Figure 3. Infiltration of FcγR⁺ Leukocytes during Neoplastic Progression in HPV16 Mice

(A) Infiltration of leukocytes expressing FcRγ (red staining, top), FcγRI (red staining, middle), and FcγRII/III (brown staining, bottom) in premalignant skin of HPV16 mice evaluated in tissue sections by immunofluorescence. Shown in the right panels is double immunofluorescence staining revealing expression of FcRγ and FcγRII/III on CD45⁺ leukocytes. Solid line, epidermal-dermal interface; e, epidermis; d, dermis; blue staining, DAPI. Scale bars represent 50 μm. (B) Differential expression of FcγRI and FcγRIII by individual leukocyte populations in premalignant skin of HPV16 (red) and HPV16/JH^−/− (blue) mice at 4 months of age. Live cells were gated as CD45⁺ leukocytes, CD45⁺CD3⁺ T lymphocytes, CD11b⁺Gr1⁺ IMCs, CD11b⁺Gr1⁻ mixed macrophages/DCs, and CD45⁺CD117⁺ mast cells. Grey line, Ig control. (C) PDSC5 tumor cells were injected as matrigel plugs into FcRγ^+/− and FcRγ^−/− mice (FVB/n). Neovascularization was evaluated by CD31 IHC (brown staining). Values reflect number of CD31⁺ vessels averaged from five high-power fields per mouse (n = 5–8 mice). Scale bars represent 25 μm. (D) Deficient tumor growth in mice lacking FcRγ. PDSC5 tumor cells were injected s.c. into FcRγ^+/− and FcRγ^−/− syngeneic FVB/n mice. Titers of IgG in serum of wild-type (WT) FVB/n versus transplanted FVB/n mice evaluated by ELISA. (C and D) Results shown are mean percentages ± SEM. Asterisks (*) indicate statistically significant differences (p < 0.05, unpaired t test).

Figure 4. FcRγ Expression Is a Critical Determinant of Squamous Carcinogenesis in HPV16 Mice

(A) Percentage of CD45⁺ cells in skin of HPV16/FcRγ^+/− and HPV16/FcRγ^−/− mice at 1, 4, and 6 months assessed by flow cytometry. (B and C) Mast cells (B, blue staining) and Gr1⁺ myeloid cells (C, brown staining) in skin of HPV16/FcRγ^+/− and HPV16/FcRγ^−/− mice at 1, 4, and 6 months of age assessed quantitatively after chloroacetate esterase histochemistry or Gr1 IHC, respectively. (D) Flow cytometric analysis of immune cell lineages as percentages of total CD45⁺ leukocytes in single-cell suspensions from nontransgenic, HPV16/FcRγ^+/−, and HPV16/FcRγ^−/− transgenic skin at 1, 4, and 6 months of age. (E) Dendritic (CD11c⁺) and macrophage (F4/80⁺) cell composition in CD11b⁺Gr1⁺ and CD11b⁺Gr1⁻ myeloid populations evaluated by flow cytometry of single cell suspensions derived from skin of negative littermate (−LM), HPV16, and HPV16/FcRγ^−/− mice at 4 months of age. (F) Attenuated angiogenesis in premalignant skin of HPV16/FcRγ^−/− mice. Density of angiogenic vasculature evaluated quantitatively by CD31 IHC (brown staining) on age-matched tissue sections. (G) Reduction in total VEGF-A and active MMP-9 protein levels in skin tissue extracts from age-matched HPV16/FcRγ^−/− and HPV16/FcRγ^+/− mice as determined by ELISA. (H) Keratinocyte proliferation evaluated as percentage of bromodeoxyuridine (BrdU)-positive keratinocytes (red staining) in ear skin tissue sections representing age-matched negative littermate (−LM), HPV16/FcRγ^+/−, and HPV16/FcRγ^−/− premalignant skin. (I) Percentages of ear skin (area) exhibiting hyperplasia by 1 month of age (Hyp) or dysplasia by 4 or 6 months of age (Dys). Values represent percentages of mice with specific neoplastic phenotypes. Statistical significance was determined using the Mann-Whitney test. Lifetime incidence of SCC was determined in HPV16/FcRγ^+/− and HPV16/FcRγ^−/− mice (97 and 132 mice/group, respectively). Tumor incidence was analyzed with the generalized Wilcoxon test. Hazard ratio was determined by Kaplan Meyer analysis of tumor incidence. All mice reflect similarly backcrossed groups at FVB/n, N5. (A–H) Results shown represent mean ± SEM (n = 5–8 mice) and asterisks (*) indicate statistically significant differences (p < 0.05, Mann-Whitney). Values represent average of five high-power fields of view per mouse. Representative images of HPV16/FcRγ^+/− and HPV16/FcRγ^−/− skin tissue sections at 4 months of age are shown. Red line, epidermal-dermal interface; e, epidermis; d, dermis. Scale bars represent 50 μm. See also Figure S3.

Figure 5. Activating FcγRs Regulate Protumor Functions of Mast Cells

(A) FcγR-dependent chemotaxis of HUVECs in response to FcγR-stimulated mast cells isolated from FcRγ^+/− or FcRγ^−/− mice. BMMCs were stimulated with 2.4G2 and anti-rat IgG (25 μg/ml; FcγR-stim.), IgG_HPV16, or IgG_wt (30 μg/ml). HUVEC migration in response to conditioned medium was assessed using a Boyden chamber assay. A specific VEGF-R2 inhibitor (DC101; 100 μg/ml) was used. HUVEC migration was quantitated by enumerating the number of migrating cells in four random fields per membrane (125× magnification). Samples were assayed in quadruplet for each assessed condition. (B) BMMCs from FcRγ^+/− or FcRγ^−/− mice were FcγR stimulated or activated with IgG_HPV16 or IgG_wt in the presence or absence of the mast cell stabilizer cromolyn (10 μM). Levels of VEGF-A in conditioned medium were assessed by ELISA. Representative analysis from three independent experiments is shown. (C) PBL migration in response to conditioned medium from FcRγ^+/− versus FcRγ^−/− BMMCs after FcγR stimulation was evaluated with a Boyden chamber assay. PBLs migrating to the lower chamber were visualized by H&E staining in five to eight random fields per well. Samples were assayed in triplicate for each tested condition. (D) FcRγ-proficient mast cells enhance tumorigenicity. PDSC5 tumor cells (blue) alone or admixed with FcRγ^+/− (red) or FcRγ^−/− (green) BMMCs were injected s.c. into FVB/n (WT) or kit^sh/sh (gray) mice at a 1:1 ratio. The asterisk (*) indicates statistically significant differences between PDSC5 cells in combination with FcRγ^+/− versus FcRγ^−/− BMMCs. The number sign (#) indicates statistically significant differences between tumor growth in syngeneic FVB/n or kit^sh/sh mice. (E) FcRγ-proficient mast cells induce angiogenesis and leukocyte infiltration of transplantable tumors. Blood vessels and leukocyte infiltration were evaluated by CD31 and Gr1 IHC. Values represent average of five high-power fields of view per tumor and six to ten tumors per category. (A–E) Data are represented as means ± SEM; *p < 0.05; **p < 0.01 (unpaired t test).

Figure 6. Tumor-Promoting Activities of CD11b⁺Gr1^+/− Myeloid Cells in HPV16 Mice

(A) Representative micrographs depicting morphology of CD11b⁺Gr1⁺ and CD11b⁺Gr1⁻ myeloid cells purified by FACS sorting from skin or spleen of HPV16 mice (4 month) and visualized via May-Grünwald-Giemsa staining (scale bars represent 50 μm). (B) Macrophages and spleen IMCs enhance tumorgenicity. Tumor growth of PDSC5 cells alone (red line) or admixed with either IMCs (blue line) or macrophages (MØ, green line; PDSC5/leukocyte = 10:1) FACS sorted from neoplastic skin (top graph) or spleen (bottom graph) of HPV16 mice at 4 months of age. Asterisks (*) and number signs (#) indicate statistically significant differences between PDSC5 cells alone and PDSC5 in combination with macrophages and IMCs, respectively. (C) Macrophages and spleen IMCs enhance tumor angiogenesis. Blood vessels in PDSC5 subcutaneous tumors were evaluated by CD31 and Gr1 IHC. Values represent average of five high-power fields of view per tumor and six to ten tumors per category. (D) FcγR-stimulated macrophages are chemotactic toward endothelial cells. Macrophages and IMCs were purified by FACS sorting from neoplastic skin and spleens of 4-month-old HPV16 mice and treated for IgG-dependent FcγR stimulation and HUVEC chemotaxis was evaluated in a Boyden chamber assay. (B–D) Data are represented as means ± SEM (*, ^#p < 0.01; **p < 0.001, unpaired t test). See also Figure S4 and Table S1.

Figure 7. FcRγ Expression Regulates Proangiogenic and Protumorigenic Properties of Macrophages

(A and B) FcRγ expression regulated macrophage protumor activity. PDSC5 tumor cells were injected alone (blue) or admixed with macrophages derived from neoplastic skin (MØ_skin) of either HPV16/FcRγ^+/− (red) or HPV16/FcRγ^−/− (green) mice (A) or instead from bone marrow-derived macrophages (MØ_BM) isolated from FcRγ^+/− (red) or FcRγ^−/− (green) mice (B). The asterisk (*) indicates statistically significant differences between PDSC5 cells admixed with FcRγ^+/− versus FcRγ^−/− macrophages. The number sign (#) indicates statistically significant differences between PDSC5 alone and PDSC5 admixed with FcRγ^−/− macrophages. Data are represented as means ± SEM; p < 0.05, unpaired t test. (C) Genes differentially expressed in macrophages isolated from neoplastic skin of HPV16/FcRγ^−/− versus HPV16/FcRγ^+/− mice. mRNA expression levels in macrophages from HPV16/FcRγ^−/− neoplastic skin (4 month old) are indicated as fold change as compared to HPV16/FcRγ^+/− macrophages with ΔCT of each gene calculated with β2-microglobulin as endogenous control. Expression was considered as statistically significantly deregulated when p < 0.05 (t test, FcRγ^−/− versus FcRγ^+/−). (D) Quantitative real-time PCR analysis of Cxcl10 and Cxcr3 mRNA expression in ear tissue from 4-month-old negative littermate (−LM), HPV16, HPV16/JH^−/−, and HPV16/FcRγ^−/− mice (n = 3–5 mice/cohort). *p < 0.05; **p < 0.01, unpaired t test. (E) VEGF₁₆₅-induced (100 ng/ml) HUVEC chemotaxis was evaluated in a Boyden chamber assay after pretreatment with recombinant CXCL10 (100 ng/ml) in the presence or absence of CXCR3 blocking antibody (10 μg/ml). *p < 0.05; **p < 0.01, unpaired t test. (F) Macrophages isolated from HPV16/FcRγ^−/− mice exhibit angiostatic activity. HUVEC chemotaxis in a Boyden chamber assay was evaluated after pretreatment with conditioned medium isolated from macrophages (purified from skin of HPV16/FcRγ^+/− or HPV16/FcRγ^−/− mice) following activation with LPS (10 ng/ml) and αCXCR3 blocking Ig (10 μg/ml). *, p < 0.05; **, p < 0.01, unpaired t test. (E and F) Pretreated HUVECs were evaluated for chemotactic migration in response to VEGF (100 ng/ml) using a Boyden chamber assay. Quantitative values reflect the number of migrating HUVECs averaged from four to five high-power fields per insert and four inserts per treatment ± SEM. At least three independent analyses were performed and one representative experiment is shown. See also Figure S5 and Table S1.

Figure 8. Activation of the FcRγ Pathway in Mast Cells and Macrophages by Humoral Immunity Regulates Squamous Carcinogenesis of HPV16 Mice

HPV16 oncogene expression initiates keratinocytes and triggers early neoplastic progression accompanied by neo- and self-antigen presentation, peripheral B cell activation/maturation, and secretion of Igs. Autoantibodies subsequently accumulate in dermal stroma of neoplastic tissue as vasculature becomes initially angiogenic and “leaky.” IgGs interact with Fcγ receptors on resident and recruited myeloid cells where they induce differential recruitment of leukocytes from peripheral blood and regulate mast cell and macrophage bioeffector functions once present in neoplastic tissue. FcRγ deficiency not only impairs the protumorigenic properties of mast cells and macrophages but also reprogram macrophages leading to enhanced angiostatic and M1 bioactivity.