Exploring the cellular plasticity and hypoxia mediated crosstalk in lung and breast tumors

Abstract

Cellular plasticity and phenotypic heterogeneity is a vital feature of solid tumors, influenced by the tumor microenvironment. Uncovering the molecular signature and functional properties of different cellular phenotypes is a critical requirement for developing advanced therapies against specific cancer. We have developed multicellular 3D spheroids using non-tumorigenic (BEAS2B), tumorigenic NSCLC (A549/NCI-H460) cells, lung fibroblasts (MRC-5), and monocytes (THP-1) to study the importance of crosstalk between cancerous and non-cancerous (stromal and immune) cells for cellular plasticity. Tumorigenic 3D spheroids show significant induction of cell proliferation, hypoxia, pluripotency markers, notable activation of cancer-associated fibroblasts, and tumor-associated macrophages. Cancer-associated fibroblasts isolated from the tumor spheroids demonstrated high glycolytic status, increased ROS levels, and altered mitochondrial health. The development of a hypoxic core is a well-known characteristic feature in advanced stage of solid tumors. In our studies, hypoxia inducing factor (HIF-1α) in the lung adenocarcinoma tissue was strongly correlated with the enhancement of miR-210-3p expression and CCL2 downregulation. Mechanistically, miR-210-3p directly binds to the 3'UTR region of the CCL2 mRNA, silences its expression, and impairs monocyte infiltration. Suppressing miR-210-3p downregulates the effect of hypoxia on CCL2 expression. Moreover, miR-210-3p inhibition in 3D tumor spheroids promotes monocyte recruitment and skewing towards anti-tumor M1 phenotype. Anti-miR-210-3p LNA delivery in lung tumor xenograft caused tumor regression, which shows miR-210-3p as a promising therapeutic. To understand the fate of macrophages within tumor spheroids, we isolated CD68+ cell populations from the late spheroids. CD68+ macrophage exhibited increased endothelial markers suggesting a possible myeloid to endothelial lineage shift. These results were validated in the zebrafish tumor xenograft model and human patient samples. There are multiple mechanisms that can contribute to cancer-associated fibroblast (CAF) heterogeneity. While investigating the involvement of the DNA damage repair pathway in the CAF heterogeneity and plasticity, it has been observed that activation of the CHK1/WEE1 pathway plays a crucial role in myoCAFs differentiation. Targeting CAFs with WEE1 inhibitor in-vivo suppresses myoCAF-rich tumor growth and promotes intratumoral CD8+ T-cell infiltration. In conclusion, the present study successfully developed the multicellular lung tumor 3D spheroids with chronic low oxygen tension in the tumor microenvironment. It investigates the cross-talk between cancerous cells and non-cancerous cells, primarily stromal and immune cells, stimulates the conversion of fibroblasts and macrophages towards the activated CAF and endothelial-like cells, respectively. Moreover, the present study also identified the role of CHK1/WEE1 inhibitors in myoCAFs differentiation and identified miR-210-3p as a promising target for immunomodulatory therapeutic strategies against lung adenocarcinoma.