Fig 2: SARS-CoV-2 infection promotes alveolar epithelial hyperplasia(A and B) Histological analysis of the alveolar epithelium stained (A) by H&E (scale bars, 200 μm) or (B) by immunofluorescence for EpCAM (yellow).(C and D) Differential gene-expression analysis (C) and GSEA using reactome (R) and hallmark (H) datasets (D) for upregulated or downregulated genes in SARS-CoV-2-infected patients compared to H1N1.(E) Histological analysis of alveolar epithelium (scale bars, 200 μm for H&E) in SARS-CoV-2 patients shows cellular hyperplasia in H&E with EpCAM+ immunofluorescent staining (yellow).(F) Differential gene-expression analysis of normal and hyperplastic alveolar epithelium in SARS-CoV-2-infected patients.(G) Heatmap representation of genes involved in alveolar epithelium proliferation (GO:0060502) and their relative expression in all SARS-CoV-2 normal alveolar epithelium, hyperplastic alveolar epithelium, and H1N1 normal alveolar epithelium (asterisk indicates significant genes between H1N1 and SARS-CoV-2 shown in the volcano plot). Differential gene expression was defined as p = 0.02 and log2 fold change of 0.5. SARS-CoV-2-infected patients (n = 3), H1N1 (n = 3), and SARS-CoV-2/H1N1 (n = 1).

Fig 3: CAF-centered heterotypic spheroids act as the MU during OC peritoneal dissemination. (A) Representative H&E staining of ascites tissues collected from HGSOC (#2, 4, and 8) and LGSOC patients (#01, 02, and 04). Bar, 50 µm. (B) The single cells from ascites were subjected to flow cytometry analyses of the percentages of these epithelial cells, leukocytes, endothelial cells, and fibroblasts. (C) Representative flow cytometry plots (top) and frequencies (bottom) showing the expression percentage of the above mentioned major resident cell categories in HGSOC and LGSOC ascites. (D) Dual immunofluorescence staining for EpCAM combined with a-SMA, PDGFRß, or prolyl 4-hydroxylase in HGSOC-derived spheroids. Bar, 100 µm. (E) Schematic illustration of peritoneal implantation of equivalent GFP-transfected SKOV3 cells and PKH-26 labeled CAFs (#1, 2, and 5). (F and G) Representative immunofluorescence image of the peritoneal heterotypic spheroids 30 min (F) and their adhesion to favored locations such as peritoneum, mesentery, and omentum 4 h after initial implantation of tumor cells and CAFs. Bar, 100 µm. (H) Immunofluorescence images showing the spreading of adhered spheroids in the peritoneal cavity 1 wk after implantation. Bars, 200 µm (left) and 50 µm (right). (I) Representative images of the newly formed metastatic nodules 2 wk after implantation. Bar, 200 µm. (J) Representative immunofluorescence images of heterotypic spheroids in murine ascites 4 wk after initial implantation. Bar, 100 µm. Data are means ± SEM and representative of four (A, C, and D) or three (F–J) independent experiments. *, P < 0.05; ***, P < 0.001, determined by Student’s t test.

Fig 4: Tumor stemness defense (TSD) phenotype can amplify the pathogen-induced bystander apoptosis (PIBA). (A) The p53 uptake assay in the culture supernatant was measured from 0 to 16 h of BCG-CM treatment in the cells. The SCC-25 SP cells were obtained as described in Figure 1 Data represent mean +/- SEM, n= 3 independent experiments. ***p < 0.0001, student t test. (B) Potential mechanism of TSD phenotype–mediated niche defense of CSCs against BCG infection. In the infected CSCs, as part of the Altruistic stemness–based (37) niche defense mechanism (6, 16, 17) HMGB1 form a complex with cytoplasmic p53 to make an, “altruistic death signal”. TLR4 internalizes the altruistic death signal, leading to induction of p53/MDM2 oscillation and activation of p53-induced pro-apoptotic genes. The EpCAM+/ABCG2+ CSCs undergoing bystander apoptosis further release the HMGB1/p53 death complex into the TME, amplifying PIBA.

Fig 5: Mdivi-1 improves the adoptive T cell therapy (ACT) in PDX tumor models by upregulating MHC-I.A Scheme of ACT therapy through tumor-specific CTLs transferred into NOD/SCID mice transplanted with autologous HNSCC or NSCLC PDXs. B Tumor volume measurements were monitored weekly after ACT for five consecutive weeks (mean ± s.e.m; n = 3 per PDX group; p < 0.0001 for both HNSCC and NSCLC; **p < 0.001 by two-way ANOVA followed by Dunnett’s tests for multiple comparisons). C Weights of harvested HNSCC or NSCLC PDXs (mean ± s.e.m; n = 3 per PDX group; p = 0.0003, 0.0006 for HNSCC and 0.0006, 0.0011 for NSCLC; *p < 0.01, **p < 0.001 by one-way ANOVA followed by Dunnett’s tests for multiple comparisons). D Apoptosis of cancer cells as determined by EpCAM and TUNEL immunostaining (mean ± s.e.m; n = 9, 3 sections per PDX; p < 0.0001 for both HNSCC and NSCLC; **p < 0.001 by one-way ANOVA followed by Dunnett’s tests for multiple comparisons). E Flow cytometric analysis of MHC-I membrane expression in isolated HNSCC and NSCLC primary cancer cells. F indicates the fold change of MFI (mean fluorescence intensity) normalized to DMSO group (mean ± s.e.m; n = 3 per PDX group; p < 0.0001 or = 0.0792, 0.3609, 0.0002 for HNSCC and 0.0002, 0.0792, 0.3855, 0.0008 for NSCLC; **p < 0.001 compared with ACT accompanied by DMSO treatment or DMSO alone by one-way ANOVA followed by Dunnett’s tests for multiple comparisons). F Representative immunofluorescence images for EpCAM and CD8 of harvested PDXs (EpCAM: red; CD8: green). DAPI, nuclear staining. Scale bars, 25 µm. G Number of IFN-?-producing CTLs as quantified by ELISpot (mean ± s.e.m; n = 3 per PDX group; p = 0.0002, 0.0004 for HNSCC and 0.0017, 0.0013 for NSCLC; *p < 0.01, **p < 0.001 by two-tailed t-test). H, I Evaluation of intracellular markers in association with cytotoxic function by flow cytometry (mean ± s.e.m; n = 3 per PDX group; p = 0.3405 or p < 0.0001 for perforin and 0.8773 or p < 0.0001 for granzyme B; **p < 0.001 compared with ACT accompanied by DMSO treatment by two-tailed t-test).