Stromal Hedgehog pathway activation by IHH suppresses lung adenocarcinoma growth and metastasis by limiting reactive oxygen species.

Activation of the Hedgehog (Hh) signaling pathway by mutations within its components drives the growth of several cancers. However, the role of Hh pathway activation in lung cancers has been controversial. Here, we demonstrate that the canonical Hh signaling pathway is activated in lung stroma by Hh ligands secreted from transformed lung epithelia. Genetic deletion of Shh, the primary Hh ligand expressed in the lung, in KrasG12D/+;Trp53fl/fl autochthonous murine lung adenocarcinoma had no effect on survival. Early abrogation of the pathway by an anti-SHH/IHH antibody 5E1 led to significantly worse survival with increased tumor and metastatic burden. Loss of IHH, another Hh ligand, by in vivo CRISPR led to more aggressive tumor growth suggesting that IHH, rather than SHH, activates the pathway in stroma to drive its tumor suppressive effects-a novel role for IHH in the lung. Tumors from mice treated with 5E1 had decreased blood vessel density and increased DNA damage suggestive of reactive oxygen species (ROS) activity. Treatment of KrasG12D/+;Trp53fl/fl mice with 5E1 and N-acetylcysteine, as a ROS scavenger, decreased tumor DNA damage, inhibited tumor growth and prolonged mouse survival. Thus, IHH induces stromal activation of the canonical Hh signaling pathway to suppress tumor growth and metastases, in part, by limiting ROS activity.


5E1 generation and purification
5E1 Hybridoma cells (Developmental Studies Hybridoma Bank) were maintained in Hybridoma medium (Gibco) with 20% FBS (heat inactivated super low IgG serum, HyClone), 1X GlutaMax (Gibco), and 2.5 mM HEPES (Fisher Scientific). Cells were maintained for 7 days and then supernatant was collected, centrifuged, and filtered through a 0.2 µm filter. Presence of 5E1 in the supernatant was verified with an ELISA assay. 3M NaCl and 0.1M Borate were added to the supernatant and pH adjusted to 8.5 and loaded on to a protein A bead column using a peristaltic pump at 1.5 mL/min at 4°C for 3 rounds. 5E1 was eluted using ImmunoPure Genetle Ag/Ab Elution Buffer (PIERCE) and then subjected to dialysis in PBS using Slide-A-Lyzer Dialysis Cassette (Thermo Fischer Scientific).

RNA in situ hybridization analysis of human lung adenocarcinoma samples
RNA In situ hybridization (ISH) was conducted by automated RNAscope assay using the Leica Bond RX autostainer (Leica Biosystems, Nussloch, GmbH) to visualize single RNA molecule per cell as a single dot in FFPE tissue samples at least. The procedure performed on BOND RX system are briefly described as the following. Tissue sections of 5um thick were deparaffinized and rehydrated, then followed with the Leica Bond prestaining and staining protocols. Antigen retrieval was performed with ER2 (BOND Epitope Retrieval Solution 2) at 95°C for 15 minutes and ACD Protease treatment at 40°C for 15 min; then RNA-specific probes were hybridized to target RNA for 120 min. The signals were amplified by multiple steps, which was followed by (Dapb) control probes were also evaluated, dapB score of <1 and PPIB score ≥2 with relatively uniform PPIB signals throughout the sample were considered adequate for analysis (data not shown).
Growth medium was replaced after 16 hours, subsequent supernatant was collected 48 hours later and centrifuged at 800xg at 4°C to precipitate cell debris. Supernatant above the cell debris pellet was collected and ultracentrifuged at 100,000xg for 2 hours. Subsequent pellet containing the lentivirus was resuspended in OptiMEM (Gibco). Titration of lentivirus was performed per previously described protocol (https://tinyurl.com/yd2qk9l6).

SURVEYOR assay
Lentivirus containing selected pSECC-Ihh was generated on a small scale (10 cm cell culture plate) using 293T cells as described above. Cell culture supernatant (containing lentivirus) was collected 72 hours after transfection and placed on 10 cm cell culture dish of 70-80% confluent Green-Go reporter cells and maintained for 48 hours. GFP+ Green-Go cells (6) were isolated by FACS. Genomic DNA from GFP+ cells was isolated using PureLink Genomic DNA Mini Kit (ThermoFischer Scientific). The sg-Ihh target region of genomic DNA was amplified by PCR using the following primers: forward 5'-GTCCCATGAGTGCTGTCGA-3' and reverse 5'-TGACCTGCATTTGCGTGGTA-3'. Mutation detection on PCR product was performed using EnGen Mutation Detection Kit (New England BioLabs) and following manufacturer instructions.

Analysis of gH2AX+ nuclei in immunohistochemical stained lung sections
Slides with immunohistochemically stained lung sections were scanned at 40x magnification using a Hamamatsu Nanozoomer. The scanned images were exported as tiff images at 10x resolution. Analysis was performed on Fiji imaging software. Tumors were outlined interactively using the custom "ROI_Draw" macro. To obtain the fraction of gH2AX positive nuclei in tumors, a custom macro, "Nuclear Fraction Calculator", was used. Briefly, "Nuclear_Fraction_Calculator" macro operates as follows. DAB positive nuclei were segmented using the "Colour Deconvolution" plugin written by Gabriel Landini (Image>Color>Colour Deconvolution in Fiji or as an ImageJ plugin available from (https://tinyurl.com/yckvnp3f). The built-in H DAB matrix is used to separate DAB staining from hematoxylin staining, the DAB channel is converted to 8-bit grayscale, the contrast is inverted, and a binary mask is generated by thresholding the image using the maximum entropy method, giving the user an option to adjust the threshold interactively. A watershed operation is used to separate connected nuclei and then ultimate points is used to represent each object with a dot. To segment the total nuclei, the original color image is converted to 8-bit grayscale and the "Mexican Hat Filter" plugin by Dimiter Prodanov (https://tinyurl.com/y7vmzrow) was used to smooth the image and enhance the edges of the nuclei. A binary mask is made, followed by watershed and ultimate points to mark each object.
The previously stored ROI set is used together with these masks for analysis of each tumor to obtain the area of the tumor, the number of DAB stained (gH2AX+) nuclei, the total number of nuclei and the fraction of DAB stained (gH2AX+) nuclei. "ROI_Draw" and "Nuclear_Fraction_Calculator" macros are compatible with ImageJ and Fiji.