Fig 2: Spib overexpression promotes lung cancer progression in subcutaneous mouse models. (A) Subcutaneous tumors in situ were excised 2 weeks after inoculation with LLC1 cells expressing empty vector or Spib. Upper panel, images of subcutaneous tumors. Lower panel, tumor weight. (B) The mice were sacrificed and lung metastases were analyzed 6 weeks after inoculation with LLC1 cells expressing empty vector or Spib (n = 5). Upper panel, representative images of lungs. Black arrow, metastatic nodules. Lower panel, quantitation of lung metastatic nodules. (C) H&E staining of lungs from mice subcutaneously injected with vector or Spib-expressing LLC1 cells (n = 5). Scale bars, 2 mm. Upper panel, representative H&E staining in lungs. Lower panel, the tumor burden was measured as the percentage of the tumor area versus the total lung area. (D) Kaplan-Meier survival curve of mice subcutaneously injected with vector or Spib-expressing LLC1 cells. Survival between LLC1 cells expressing empty vector and LLC1 cells expressing Spib was significantly different (P < 0.05; n = 6, vector; and n = 5, Spib). (E) CMT167 cells expressing control or shRNA against Spib were subcutaneously injected into C57BL/6 mice. Two weeks later, subcutaneous tumors were removed. Left, images of subcutaneous tumors. Right, tumor weight. Mean ± SD. *P < 0.05. **P < 0.01. ***P < 0.001.

Fig 3: Spib overexpression increases TAM infiltration in subcutaneous mouse models. (A) Representative flow cytometry profiles and percentages of TAMs in CD45+ TILs in subcutaneous tumors. n = 3-4. (B) Representative flow cytometry profiles and percentages of CD206+ cells in CD11b+ F4/80+ cell populations in subcutaneous tumors. n = 3. (C) Quantification of Tregs in CD45+ TILs in subcutaneous tumors by flow cytometry. n = 3-4. (D) Quantification of monocytic myeloid-derived suppressor cells (M-MDSCs) and granulocytic myeloid-derived suppressor cells (G-MDSCs) in CD45+ TILs in subcutaneous tumors by flow cytometry. n = 3-4. (E) Quantification of CD8+ T cells and CD4+ T cells in CD45+ TILs in subcutaneous tumors by flow cytometry. n = 3-4. (F) Quantification of NK cells in CD45+ TILs in subcutaneous tumors by flow cytometry. n = 3-4. (G) Immunostaining with anti-CD163 was performed in subcutaneous tumors derived from LLC1 cells expressing Spib or empty vector. Scale bars, 20 µm. Left, representative pictures of CD163 staining. Right, quantified CD163 expression in subcutaneous tumors. FoV = field of view. n = 3 mice for each group. (H) Immunostaining with anti-CD8 was performed in subcutaneous tumors derived from LLC1 cells expressing Spib or empty vector. Scale bars, 20 µm. Left, representative pictures of CD8 staining. Right, quantified CD8 expression in subcutaneous tumors. n = 3 mice for each group. (I, J) CD11b+ cells purified from subcutaneous tumors derived from LLC1 cells expressing Spib or empty vector were cocultured with T cells for 2 days. Quantification of IFN-?+ in CD8+ T cells by flow cytometry (I). Quantification of the geometric mean fluorescence intensity of granzyme B in CD8+ T cells (J). n= 3. (K) In vitro suppressive activity of tumor-infiltrating CD11b+ cells purified from subcutaneous tumors derived from LLC1 cells expressing Spib or empty vector. Representative histograms of CD8+ T cell proliferation (left panel). Quantification of CD8+ T cell proliferation using carboxyfluorescein succinimidyl ester (CFSE) (right panel). n = 3. Mean ± SD. *P < 0.05. **P < 0.01. ***P < 0.001.

Fig 4: Spi-B expression positively correlates with the proportion of CD163+ macrophages in human lung cancer tissues. (A) Immunostaining of human CD163 and Spi-B in paraffin-embedded lung tissues using phase-contrast microscopy (400×). Representative images show a lack of Spi-B staining in normal lung epithelial cells (a), but positive Spi-B staining in lymphocytes (b) and positive CD163 staining in alveolar macrophages (c). In lung cancer tissues, CD163-expressing TAMs surrounded and infiltrated cancer masses of strong Spi-B staining (d, e). Scale bars, 20 µm. (B) Semiquantitative scoring was performed. CD163 scores were associated with Spi-B scores (r = 0.6098, P < 0.0001). (C) Kaplan-Meier survival rates for 73 subjects with low (staining scores = 3, n = 47, blue line) versus high (staining scores > 3, n = 26, red line) CD163 expression were compared (P = 0.0307). (D) Kaplan-Meier survival rates for 73 subjects with low (staining scores < 4, n = 29, blue line) versus high (staining scores = 4, n = 44, red line) Spi-B expression were compared (P = 0.0366).

Fig 5: SPIB upregulates the expression of TAM polarization- and recruitment-related genes. (A) GSEA of C5 signatures (positive regulation of macrophage activation, macrophage differentiation, and regulation of monocyte chemotaxis) in microarray data of A549 cells expressing SPIB and control vector. (B–E) Validation of microarray data by RT-qPCR. RNA was purified from A549 cells expressing SPIB or empty vector (B), H1299 cells expressing SPIB or empty vector (C), LLC1 cells expressing Spib or empty vector (D), and CMT167 cells with knockdown of Spib or control (E). Mean ± SD. *P < 0.05. **P < 0.01 ***P < 0.001.