Microenvironment-derived ADAM28 prevents cancer dissemination

Abstract

Previous studies have linked cancer cell-associated ADAM28 expression with tumor progression and metastatic dissemination. However, the role of host-derived ADAM28 in cancer dissemination processes remains unclear. Genetically engineered-mice fully deficient for ADAM28 unexpectedly display increased lung colonization by pulmonary, melanoma or breast tumor cells. In experimental tumor cell dissemination models, host ADAM28 deficiency is further associated with a decreased lung infiltration by CD8⁺ T lymphocytes. Notably, naive ADAM28-deficient mice already display a drastic reduction of CD8⁺ T cells in spleen which is further observed in lungs. Interestingly, ex vivo CD8⁺ T cell characterization revealed that ADAM28-deficiency does not impact proliferation, migration nor activation of CD8⁺ T cells. Our data highlight a functional role of ADAM28 in T cell mobilization and point to an unexpected protective role for host ADAM28 against metastasis.

Keywords: ADAM28; CD8+; T lymphocytes; lung; metastasis.

Figure 1. Knockdown strategy of the gene encoding ADAM28 in mouse

(A) Targeting strategy to generate ADAM28 floxed (targeted allele) and ADAM28 null (KO allele) alleles. Restriction map depicting the wild-type (WT) ADAM28 gene locus (exons 1-6 (represented by white boxes)). In the targeting vector, the exon 2 has been flanked by loxP sites (grey triangles) and a neomycin selection cassette (NEO) flanked by frt sites (black ellipses). A diphteria toxin A (DTA) cassette is inserted at the 5′-site of the targeting vector. The grey bars under the restriction maps indicate sizes of BamHI (B) and HindIII (H) digestion–derived restriction fragments detected in wild-type and recombinant loci. DNA probes for Southern blot analysis (grey boxes) were designed outside the targeting vector. The ADAM28 null allele (KO) is obtained after a first Flp-excision of the NEO cassette followed by a Cre-mediated excision of exon 2. (B) DNA genotyping of WT (lanes 1-2) and ADAM28 KO littermates (lanes 3-4). ADAM28 KO mice bear a 153 bp-DNA short copy, while WT mice bear a 623-bp DNA copy of the ADAM28 gene. (C) Semi-quantitative RT-PCR measuring ADAM28 transcripts in lungs of tumor-free WT (lanes 1-3) and ADAM28 KO (lanes 4-6) mice. 28S ribosomal RNA levels are shown as loading control. (D) Quantification of relative ADAM28 mRNA expression in lungs of WT and ADAM28 KO mice. Results are expressed as arbitrary units corresponding to the ADAM28/28S ratio. Bars represent SEM (n=3). (E) Semi-quantitative RT-PCR measuring ADAM28 mRNA transcripts in thymus and lungs of WT (lanes 1-3; 7-9) and ADAM28 KO (lanes 4-6; 10-12) mice using primers targeting exon 2. (F) Wild-type and ADAM28 knockout littermates at 8 weeks of age. (G) Weight-gain curves of WT and ADAM28 KO littermates. Bars represent SEM. (H) Number of matings and mean litter size ± SEM for each genotype. These values were determined by considering the number of births obtained over one year for one couple per genotype.

Figure 2. ADAM28 deletion in host tissues promotes metastasis to lung tissues

(A) Biophotonic monitoring of lungs of WT (n=18) and ADAM28 KO mice (n=22), 21 days after intravenous injection of luciferase- transfected LLC cells. (Mann-Whitney; p=0.2). Images shown have been taken in ‘Photon mode’. The right panel shows the quantification of luminescent signals in regions of interest (ROI) in lungs. (B) Quantification of tumor density on HE-stained lung sections from WT (n=25) and ADAM28 KO (n=20) mice at day 21 after intravenous LLC cell injection. (Mann-Whitney; ^*p< 0.05). Representative data from three independent experiments are shown. (C) Representative images of HE-stained lungs from WT and KO mice bearing LLC tumors (black arrows) (scale bar: left panel: 2500 μm). The right panel (scale bar: 250 μm) is a zoom of a tumor area present in lungs of each genotype. (D) Kaplan-Meier curves measuring survival of WT (n=13) and ADAM28 KO (n=17) mice over 50 days after intravenous LLC cell injection (Mantel-Cox; ^*p=0.022). (E) Quantification of tumor density in HE-stained lung tissue slides of WT (n=20) and ADAM28 KO (n=19) mice at day 28 after B16K1 cell injection. (Mann-Whitney; ^**p< 0.01). Representative data from three independent experiments are shown. (F) HE-stained lung sections from WT and KO mice bearing B16K1 tumors (arrow) (scale bar: 2500 μm) (G) Quantification of tumor density on HE-stained lung sections from WT (n=11) and ADAM28 KO (n=11) mice at day 14 after 4T1 cell injection. (Student's t test; ^*p<0.05). Representative data from two independent experiments are shown. (H) Representative images of HE-stained lungs from WT and KO mice bearing 4T1 tumors (black arrows) (scale bar: 2500 μm).

Figure 3. ADAM28 deficiency affects CD8⁺ T cell mobilization to splenic and pulmonary tissues in tumor-bearing mice

(A-B) Percentages of CD8⁺/CD3⁺ T cell populations were evaluated in spleen (WT=11, KO=13) and lung tissues (WT=18, KO=21) from WT and ADAM28 KO mice 21 days following intravenous LLC cell injection (Student's t test; Mann-Whitney; spleen: ^*p <0.05; lung: ^**p <0.01). (C) Representative images of CD8⁺ T cells stained with anti-CD8 antibody (green) present in lungs bearing equally- sized tumors from WT and ADAM28 KO mice. Nuclei were stained with DAPI (blue). Scale bar: 250 μm. (D) Density of CD8 staining was measured and reported to total lung area of WT (n = 11) and ADAM28 KO (n = 15) mice (Mann-Whitney; ^*p <0.05). (E) Evaluation of CD4⁺/CD3⁺ T cell population percentages by flow cytometry in lungs of WT (n=23) and ADAM28 KO (n=27) mice 21 days following intravenous LLC cell injection. (F) Evaluation of Treg (FoxP3⁺/CD4⁺) percentages in lungs of WT (n=12) and ADAM28 KO (n=15) mice at day 21 post-injection of LLC cells. (G) Percentages of B cells (B220⁺/CD3⁻) infiltrated within lungs of WT (n=10) and KO (n=14) mice 21 days after LLC cell injection. (H-I) Percentages of CD8⁺ T cells evaluated by flow cytometry on spleen and lungs from WT and KO (n = 8) at day 35 after B16K1 cell injection (spleen: Student's t test; ^*p < 0.05) (lung: Mann-Whitney; ^**p < 0.01). (J-K) Flow cytometry analysis performed on spleen and lungs from BALB/cJRj WT (n=11) and ADAM28 KO (n=11) mice 14 days following intravenous 4T1 cell injection. (Student's t test; spleen: ^*p < 0.05; lung: ^***p < 0.001).

Figure 4. ADAM28 deficiency reduces CD8⁺ T cell mobilization to splenic and pulmonary tissues in tumor-free mice

(A-B) CD8⁺/CD3⁺ T cell percentages assessed in spleen (n=16) and lungs (WT=9, KO=10) of tumor-free C57BL/6JRj WT and ADAM28 KO mice (Student's t test; ^***p < 0.001). (C-D) Flow cytometry analyses were performed to evaluate CD8⁺/CD3⁺ cell populations present in spleen (WT=6, KO=5) and lungs (WT=5, KO=5) of tumor-free BALB/cJRj WT and ADAM28 KO mice (Mann-Whitney; ^**p <0.01). (E) Percentages of CD8⁺ T cells (CD8⁺/CD4⁻ B220⁻) evaluated by flow cytometry in thymus of tumor-free WT (n=11) and ADAM28 KO (n=11) mice (Student's t test; p>0.05). (F) Representative HE-stained sections of thymus derived from WT and ADAM28 KO mice. Cortex (C) and medulla (M). Scale bar: 500 μm. (G) Thymus weight of 6 week-old WT (n=5) and ADAM28 KO (n= 5) mice (Student's t test).

Figure 5. Functional features of ADAM28 deficient CD8⁺ T cells are not impaired

(A-C) Production of KC (A), CXCL10 (B) and IL-12 (C) was assessed by ELISA on lung protein extracts from WT (n=11) and ADAM28 KO (n=16) mice bearing equally-sized tumors (Student's t test; Mann-Whitney; p>0.05). (D-F). Quantification of Granzyme B (GrzB) (D), CXCR3 (E) and IFN-γ (F) expression on CD8⁺ T cells by flow cytometry in total lung extracts of WT (n=6) and ADAM28 KO (n=8) mice after intravenous LLC cell injection (Day 21) (Student's t test; ^**p < 0.01). (G) Proliferation assay performed on ADAM28 KO (n=6) and WT (n=6) CD8⁺ T cells stimulated by CM from tumor cells (LLC, 4T1, B16K1) or by CM from CD8⁺ T cells (Mann-Whitney). (H) Migration assay on CD8⁺ T cells isolated from WT (n=4) and ADAM28 KO (n=4) mice. Chemoattractants used in the lower chamber of the Boyden Chamber were either CXCL10, CM from tumor cells (LLC, 4T1, B16K1) or CM from CD8⁺ T cells previously isolated from WT or ADAM28 deficient mice (Mann-Whitney). All results were normalized to the migration of WT CD8⁺ T cells (without stimulation) considered as baseline migration and expressed as fold increase from baseline.