(M1230-04-22) Effect of Lung Cancer Exosomal Galectin-3 Binding Protein on Bone Metastasis

Purpose: Around 30-40% of lung cancer (LC) metastasis results in skeletal metastasis with abnormal stimulation of bone-resorbing cells, i.e., osteoclasts (OC) [1]. This phenomenon disrupts the bone homeostatic environment, weakening skeletal structures, bone loss, and life-threatening fractures. Overall, treatment strategies provide a temporary fix but do not significantly improve the patient’s condition. A deeper understanding of the LC pathology is necessary to counter osteolytic metastasis with significant outcomes, i.e., investigating the signaling proteins secreted by LC cells. LC metastasis to the bone is enabled by the secretion of various biomolecules alone or enveloped in exosomes, altering the recipient cell's phenotypic and genotypic characteristics. This study focuses on the exosomal galectin-3-binding protein (Mac-2-BP). This specific glycoprotein binds to various ligands such as galectin 3 (Mac-2) present either on the cell surface or intracellularly and activates a myriad of signaling pathways resulting in abnormal cell division, dislodging of cells to aid in the invasion, metastasis, and evading immune cells [2]. Mac-2-BP resembles a free-floating receptor that instrumentally facilitates the aggressiveness and invasive nature of breast, prostate, and pancreatic cancers, but its role in lung cancer remains unknown. This study will investigate LC-derived exosomal Mac-2-BP’s role in osteolytic metastasis.

Methods: Using LC cells of different metastatic natures (A549, H1437, SK MES-1 & CALU-6), the effect of LC secretomes (conditioned media and exosomes) was assessed on pre-OC (RAW 264.7) cell viability. OC differentiation and function via pit resorption assay were monitored after co-incubation of RAW 264.7 with LC cells or treatment with LC cell secretomes. The extent of OC differentiation was based on producing a cytochemical marker, tartrate-resistant acid phosphatase (TRAP), and western blotting to analyze the expression of key markers such as NFAT2, RANKL, and cFos. Further, exosomes from LC cells were isolated, characterized, lysed, and extracted for their proteinous content via gel digestion for Liquid chromatography/Mass spectrometry (LC/MS) analysis, using BEAS-2b (lung epithelial cells) derived exosomes as a negative control. Proteomic profiling and western blotting analysis investigated the expression and involvement of Mac-2-BP and Mac-2 in LC exosomes. In addition, LC cells with differential expressions of Mac-2-BP and Mac-2 were injected intravenously into athymic mice while monitoring skeletal parameters over 80 days using DEXA.

Results: LC cell secretomes did not hinder pre-OC cell viability; in fact, we observed a significant increase in cell viability upon exposure to LC secretomes of H1437, A549, and Calu- 6 (p < 0.01) compared to the negative control. Treatment with LC cells indirectly via secretomes did not exhibit any significant changes in TRAP activity or OC count; however, an increase in the number of OCs with nuclei greater than 10 compared in wells treated with A549, H1437, and SK MES-1 to the positive control (Fig 1). This trend was reproduced when assessing the effect and function of differentiating OCs co-incubated with LC cells. A significant increase (p < 0.05) in the number of osteoclastic pits was observed from OCs co-incubated with A549 compared to the positive control. Western blotting analysis revealed an upregulation in the NFAT2 and cFos expression in OCs co-cultured with A549 and SK MES-1, indicating LC-derived signaling proteins targeting crucial OC differentiation markers. In addition, RANKL expression was observed to be upregulated in OCs co-incubated with A549 cells, like the positive control (Fig 2C). Mac-2-BP expression was robust in exosomes derived from A549, SK MES-1, and H1437. In contrast, Mac-2-BP expression was strong in lysates from H1437 and SK MES-1 but low in A549. While Mac-2 expression was expressed strongly in H1437, SK MES-1, and Calu-6 (Fig 2 A & B). It was also observed that Mac-2-BP molecular weight in OCs decreased to ~66 kDa when compared to exosomal Mac-2-BP at ~116 kDa. These findings suggest that Mac-2-BP underwent proteolytic cleavage upon complementary binding to exert its effect. DEXA analysis revealed that intravenous administration of LC cells to athymic mice after 80 days exhibited no significant changes in body weight, bone mineral density, or content (Fig 3), suggesting that intratibial administration of LC cells would be a better model to induce osteolytic lesions for clinical correlation.

Conclusion: LC cells increase OC size resulting in larger resorption sites. LC-derived exosomes confirmed a high expression level of Mac-2-BP, whereas its respective cell lysates exhibited a high level of Mac-2 expression. In addition, OC markers such as NFAT2 and cFos are upregulated in OCs co-incubated with LC cells, especially A549, suggesting that Mac-2-BP binds to Mac-2 and targets OC markers such as NFAT2 and cFos for increased OC differentiation and activation. Overall, the interaction of LC-derived Mac-2-BP and Mac-2 most likely facilitated OC differentiation and function, which are the main features of osteolytic bone metastasis.

References: 1. Wu, S., et al., Current progress and mechanisms of bone metastasis in lung cancer: a narrative review. Translational Lung Cancer Research, 2021. 10(1): p. 439.
2. Capone, E., S. Iacobelli, and G. Sala, Role of galectin 3 binding protein in cancer progression: a potential novel therapeutic target. Journal of Translational Medicine, 2021. 19(1): p. 405.

Acknowledgements:This work was supported by NEOMED Foundation

Figure 1: Effect of Pre-OC cells co-incubated with LC cells on OC differentiation. Pre-osteoclastic (RAW 264.7) cells were cultured and differentiated to OCs with growth media containing RANKL (30 ng/mL) (positive control). Cells were co-incubated with different LC cells; A549, H1437, SK MES-1, and Calu-6 together with RANKL. The extent of OC differentiation was assessed by TRAP activity, (A) the number of TRAP stained (pink) multinucleated OCs containing nuclei 3-10 and greater than 10, and (B) the extent of TRAP staining (pink coloration). Each data point represents mean ± SD; n=4, #p < 0.05 versus positive control (RANKL).

Figure 2: Western Blot analysis of Mac-2-BP, Mac-2, and OC differentiation markers. The expression of Mac-2-BP and Mac-2 were assessed in (A) LC-derived exosome and (B) LC cell using Ponceau stain and GAPDH as loading controls for exosomes and cell lysates, respectively, with BEAS-2b as the negative control. (B) The expression of OC markers (NFAT2, cFos, and RANKL) and Mac-2-BP expression were assessed in differentiated OCs using beta-actin as the loading control. RAW 264.7 cells were differentiated into OCs with treatment with RANKL alone (positive control) or RANKL co-cultured with LC cells.

Figure 3: DEXA analysis of 80-day post-LC cell intravenous administration in athymic mice. (A) Representative DEXA image of nude mice injected with LC cell via tail vein followed by (B) Bone Mineral Density and (C) Bone Mineral Content analysis of all athymic mice with LC cells administered. Mice without LC cells acted as control.