Poster S14 - Senescence-Associated Modifications in the Fibrotic Lung Niche for Breast Cancer Metastasis

Introduction: Older breast cancer patients treated with radiotherapy suffer from increased incidence of radiation induced lung injury and metastasis; these patients also have more senescent cells, which accumulate with age and after radiation treatment1–3. Though senescence is a critical mechanism of arresting damaged cells from proliferating, senescent cells are metabolically active and communicate with other cells directly and by transferring pro-inflammatory molecules in the senescence associated secretory phenotype (SASP). Consequently, senescent cells are critical in recruiting immune cells and mediating extracellular matrix (ECM) remodeling in aging and diseased tissues. In fact, senescent cells and their pro-inflammatory SASP have been described as key mediators of pulmonary fibrosis, a common side effect of breast cancer radiotherapy. We hypothesize that ECM remodeling by senescent cells creates fibrotic niches or sites for cancer metastasis to the lungs. The fact that breast cancer metastasis to the lungs is correlated with increasing age is supportive of this hypothesis4. However, major gaps still remain in our understanding of how aging and radiation-induced senescence affect pathological ECM remodeling and metastasis in the lungs. Our study is investigating the hypothesis that ECM remodeling by senescent cells creates fibrotic niches or sites for cancer metastasis to the lungs.

Materials and

Methods: Fibroblast-Derived Matrix (FDM). Immortalized human lung fibroblasts (LF1) were used to generate FDM and were maintained at 37° C and 2.5% oxygen (hypoxia). To induce senescence, cells were treated with 15 Gy ionizing radiation using the Mark-I Cesium Irradiator. Cells were then cultured for 7-21 days on gelatin coated glass coverslips in ECM-promoting media to induce matrix production before second harmonic generation imaging of collagen structure with an Olympus Multiphoton Microscope. An in-house MATLAB code was used to quantify the spatial collagen organization from gridded images to look at local and global ECM remodeling near ECM-embedded cells and throughout the sample.
Senescence Characterization. We distinguished senescence based on multiple factors, including reduced proliferation (BrdU incorporation), increased SA-β-gal expression, and increased expression of SASP factors (IL-6, IL-8, IL-1β), and senescence markers, including cyclin dependent kinases p16 and p21 using qPCR.
Precision Cut Lung Slices (PCLSs) were generated from fresh lung tissue from C57BL/6 mice. Lungs were injected with agarose to inflate them before cutting into slices using a vibrating microtome. PCLSs were transferred to tissue culture plates and maintained at 37° C and 5% carbon dioxide in normal growth media for 7-14 days in 2% oxygen (hypoxia) or ambient oxygen (normoxia). PCLS were treated on day 2 with 15 Gy ionizing radiation to induce senescence. At the end of the growth period, tissues were fixed and stained for senescent cells (P16) and collagen fibers (F-CHP), and cytoskeletal protein (Phalloidin for F-actin)before imaging with Olympus Yokogawa Spinning Disk Confocal Microscope.

Results, Conclusions, and Discussions: This study used an in vitro fibroblast derived matrix model, human lung tissues, and precision cut lung slices (PCLSs) formed from mouse tissues to look at senescence-associated ECM remodeling. We developed a fibroblast derived matrix model to directly examine ECM remodeling by senescent cells; our results show senescence is associated with increased deposition of highly disordered collagen (Fig. 1A-C), which was correlated with highly heterogeneous actin filament structure and the formation of multi-lateral tensions. Similar abnormal ECM structure was seen in fibrotic lung from older patients with more p16 positive senescent cells (Fig. 1D-E). To understand the effect of radiation induced accumulation of senescent cells in vivo tissue microenvironment, we irradiated PCLS tissues from mouse lungs and maintained them for two weeks. We identified senescent cells using P16 and the surrounding matrix architecture using collagen binding peptide F-CHP. We also found that irradiated PCLS from mouse showed a higher number of P16 positive cells and displayed a higher degree of disorganized cellular actin and surrounding collagen structure (Fig. 1F).
In vivo percentage of senescent cells is very low (before radiation < 1%)5, making it difficult to identify them. However, their ECM remodeling phenotype may promote malignant cancer cell behavior. Thus, we anticipate that senescent cells will reside near metastatic cells making it easier to identify them. The ECM remodeling phenotype may promote adhesion and spreading of cancer cells through increased integrin expression and tissue stiffening; whereas, soluble factors in the SASP may contribute to increased proliferation and motility of breast cancer cells. Thus, we expect to find larger metastatic foci in irradiated lung tissues and a larger percentage of senescent cells.

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