For traditional laboratory microscopy observation, the multi-dimensional, real-time, in situ observation of three-dimensional (3D) tumor spheroids has always been the pain point in cell spheroid observation. In this study, we designed a side-view observation petri dish/device that reflects light, enabling in situ observation of the 3D morphology of cell spheroids using conventional inverted laboratory microscopes. We used a 3D-printed handle and frame to support a first-surface mirror, positioning the device within a cell culture petri dish to image cell spheroid samples. The imaging conditions, such as the distance between the mirror and the 3D spheroids, the light source, and the impact of the culture medium, were systematically studied to validate the in situ side-view observation. The results proved that placing the surface mirror adjacent to the spheroids enables non-destructive in situ real-time tracking of tumor spheroid formation, migration, and fusion dynamics. The correlation between spheroid thickness and dark core appearance under light microscopy and the therapeutic effects of chemotherapy doxorubicin and natural killer cells on spheroids’ 3D structure was investigated.

Expression of NPRL2/TUSC4, a tumor-suppressor gene, is reduced in many cancers including NSCLC. Restoration of NPRL2 induces DNA damage, apoptosis, and cell-cycle arrest. We investigated NPRL2 antitumor immune responses in aPD1^R/KRAS/STK11^mt NSCLC in humanized-mice. Humanized-mice were generated by transplanting fresh human cord blood-derived CD34 stem cells into sub-lethally irradiated NSG mice. Lung-metastases were developed from KRAS/STK11^mt/aPD1^R A549 cells and treated with NPRL2 w/wo pembrolizumab. NPRL2-treatment reduced lung metastases significantly, whereas pembrolizumab was ineffective. Antitumor effect was greater in humanized than non-humanized-mice. NPRL2 + pembrolizumab was not synergistic in KRAS/STK11^mt/aPD1^R tumors but was synergistic in KRAS^wt/aPD1^S H1299. NPRL2 also showed a significant antitumor effect on KRAS^mt/aPD1^R LLC2 syngeneic-tumors. The antitumor effect was correlated with increased infiltration of human cytotoxic-T, HLA-DR⁺DC, CD11c⁺DC, and downregulation of myeloid and regulatory-T cells in TME. Antitumor effect was abolished upon in-vivo depletion of CD8-T, macrophages, and CD4-T cells whereas remained unaffected upon NK-cell depletion. A distinctive protein-expression profile was found after NPRL2 treatment. IFNγ, CD8b, and TBX21 associated with T-cell functions were significantly increased, whereas FOXP3, TGFB1/B2, and IL-10RA were strongly inhibited by NPRL2. A list of T-cell co-inhibitory molecules was also downregulated. Restoration of NPRL2 exhibited significantly slower tumor growth in humanized-mice, which was associated with increased presence of human cytotoxic-T, and DC and decreased percentage of Treg, MDSC, and TAM in TME. NPRL2-stable cells showed a substantial increase in colony-formation inhibition and heightened sensitivity to carboplatin. Stable-expression of NPRL2 resulted in the downregulation of MAPK and AKT-mTOR signaling. Taken-together, NPRL2 gene-therapy induces antitumor activity on KRAS/STK11^mt/aPD1^R tumors through DC-mediated antigen-presentation and cytotoxic immune-cell activation.