Treatment of late-stage lung cancers remains challenging with a five-year survival rate of 8%. Immune checkpoint blockers (ICBs) revolutionized the treatment of non-small cell lung cancer (NSCLC) by reactivating anti-tumor immunity. Despite achieving durable responses, ICBs are effective in only 20% of patients due to immune resistance. Therefore, synergistic combinatorial approaches that overcome immune resistance are currently under investigation. Herein, we studied the immunomodulatory role of Withaferin A (WFA)—a herbal compound—and its effectiveness in combination with an ICB for the treatment of NSCLC. Our in vitro results show that WFA induces immunogenic cell death (ICD) in NSCLC cell lines and increases expression of the programmed death ligand-1 (PD-L1). The administration of N-acetyl cysteine (NAC), a reactive oxygen species (ROS) scavenger, abrogated WFA-induced ICD and PD-L1 upregulation, suggesting the involvement of ROS in this process. Further, we found that a combination of WFA and α-PD-L1 significantly reduced tumor growth in an immunocompetent tumor model. Our results showed that WFA increases CD-8 T-cells and reduces immunosuppressive cells infiltrating the tumor microenvironment. Administration of NAC partially inhibited the anti-tumor response of the combination regimen. In conclusion, our results demonstrate that WFA sensitizes NSCLC to α-PD-L1 in part via activation of ROS.

Alterations to NPRA signaling may lead to inhibition of cancer stem cell (CSC) proliferation; we report the development of a 3D tumor/stroma coculture model to study and further define the role that NPRA plays in CSC development and drug resistance. Objectives: Determine changes in cell phenotype caused by culture on a 3D nanofiber scaffold or suspension culture, whether these changes represent an enrichment of CSCs, the mechanism of enrichment/role of NPRA. Examine the roles of stromal cell types in tumor growth, CSC development, and drug resistance in an in vitro coculture. Assess CSC expansion and drug resistance in 3D scaffold tumoroids or suspension with/without cancer associated fibroblasts (CAF) as well as in isolated CSC populations. Methods: CSCs were assayed by flow cytometry (CD44+/CD24-, CD133+) and isolated by FACS or MACS. Stemness was assayed by SOX2 OCT4 NANOG expression and aldehyde dehydrogenase(ALDH) activity with the ALDEFLUOR kit. Sphere forming assays (SFAs) for anchorage independent growth with/without NPRA inhibitors and/or stromal cells were performed and measured by bright field microscopy. Tumor initiation was assayed in the flanks of syngeneic or NSG mice. NPRA KO mice were used to determine the influence of stromal NPRA on tumor growth. Cancer cells were grown on 3D nanofiber scaffold with/without stromal cells as an in vitro model of the tumor microenvironment. CSC enrichment was assayed by CSC marker expression and SFAs. NPRA knockout cell lines were made using CRISPR/Cas9, verified by flow cytometry and qPCR. Knockout cell lines were tested for their tumor initiation, CSC marker expression, and SFA in the presence or absence of stromal cells. Results: Introduction of factors secreted by CAF increased the tumor cells' ability for attachment-independent growth; however, this was not seen with NPRA inhibitor treatment. Putative CSCs collected from tumor spheres initiated tumors in mice at lower cell numbers than their parental cell lines. 3D culture of tumor biopsies increased expression of CD44+ CD24- ALDH+ compared to primary tumor or monolayer. 3D culture increased the drug resistance of cancer cells, compared to 2D. Cells from the CD44+CD24- population initiated tumors at lower numbers than parental. Tumor spheres initiated in vivo tumors at lower cell counts than monolayer cultures. These tumors grew faster in wild type mice vs NPRA KO mice. CRISPR genome editing allowed us to create NPRA knockout cell lines to confirm the results of the inhibitor studies. Coculture of different cell types with/without NPRA KO determined NPRA's influence on CAF supporting tumor growth. Conclusions: The 3D scaffold offers a cell culture environment causing cancer cells to retain more in vivo behavior. This model can increase the CSC phenotype and can be used to study the mechanisms of CSC proliferation, drug resistance, and the role of stromal cells. We hypothesized that NPRA signaling is key for this process and we found that inhibiting NPRA signaling reduces the ability for attachment-independent growth even in the presence of tumor supporting CAF and their in vivo tumor initiation ability. Cancer cell growth was reduced by NPRA inhibition and NPRA signaling is important for the initiation and maintenance of tumors. We plan to use the scaffold model to determine NPRA signaling’s influences on CSC development and its effects on each stromal cell type within tumors. Citation Format: Ryan J. Green, Mark Howell, Rajesh Nair, Shruti Padhee, Scott Antonia, Eva Samal, Jit Banerjee, Shyam Mohapatra, Subhra Mohapatra. NPRA signaling in the tumor/stroma microenvironment influences the growth of stem-like cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A066.