Role of macrophages in cancer progression and targeted immunotherapies.

The vast complexity of the tumor microenvironment (TME) aggrandizes the underlying principles responsible for immune escape, therapy resistance, and treatment failure. The stromal and immune cell population circumjacent to the tumor cells affects the cancer cell cycle leading to tumor progression. Tumor-associated macrophages (TAMs) exhibiting a unique M2 polarization state constitute a significant portion of the TME. They serve as tumor suppressors at early stages and tumor promoters at advanced stages by governing various microenvironmental cues. TAMs secreted various pro-tumoral cytokines, chemokines, and matrix metalloproteases are known to regulate the different cell cycle molecules including checkpoint inhibitors in cancer cells leading to cell cycle progression with faulty cellular components. Moreover, TAMs are well-known immunosuppressors and thereby facilitating the tumor cells' evasion from immune recognition. This chapter will describe the interaction between TAMs and tumor cells, the involvement of TAMs in the regulation of cancer cell progression by controlling cell cycle checkpoints or molecular pathways, and current TAM-based therapies, including restriction of TAM recruitment, anti-survival strategies, or switching polarity. Moreover, this chapter will also emphasize recently developed drug targets and CAR-macrophage cell therapy that restricts tumor progression.

Anti-cancer drug molecules targeting cancer cell cycle and proliferation.

Cancer, a vicious clinical burden that potentiates maximum fatality for humankind, arises due to unregulated excessive cell division and proliferation through an eccentric expression of cell cycle regulator proteins. A set of evolutionarily conserved machinery controls the cell cycle in an extremely precise manner so that a cell that went through the cycle can produce a genetically identical copy. To achieve perfection, several checkpoints were placed in the cycle for surveillance; so, errors during the division were rectified by the repair strategies. However, irreparable damage leads to exit from the cell cycle and induces programmed cell death. In comparison to a normal cell, cancer cells facilitate the constitutive activation of many dormant proteins and impede negative regulators of the checkpoint. Extensive studies in the last few decades on cell division and proliferation of cancer cells elucidate the molecular mechanism of the cell-cycle regulators that are often targeted for the development of anti-cancer therapy. Each phase of the cell cycle has been regulated by a unique set of proteins including master regulators Cyclins, and CDKs, along with the accessory proteins such as CKI, Cdc25, error-responsive proteins, and various kinase proteins mainly WEE1 kinases, Polo-like kinases, and Aurora kinases that control cell division. Here in this chapter, we have analytically discussed the role of cell cycle regulators and proliferation factors in cancer progression and the rationale of using various cell cycle-targeting drug molecules as anti-cancer therapy.

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids.

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Development of a Multicellular 3D Tumor Model to Study Cellular Heterogeneity and Plasticity in NSCLC Tumor Microenvironment.

Heterogeneity is a characteristic feature of solid tumors. Intra-tumor heterogeneity includes phenotypic diversity, epigenetic abnormalities, cell proliferation, and plasticity that eventually drives disease progression. Studying tumor heterogeneity in 2D culture is challenging as it cannot simulate the microenvironmental features, such as hypoxia, nutrient unavailability, and cell-ECM interactions. We propose the development of multicellular (tri-culture) 3D spheroids using a hanging drop method to study the non-tumorigenic (BEAS-2B) vs. tumorigenic NSCLC (A549/NCI-H460)cells' interaction with lung fibroblasts (MRC-5) and monocytes (THP-1). Unlike the non-tumorigenic model, the tumorigenic 3D spheroids show significant induction of cell proliferation, hypoxia, pluripotency markers, notable activation of cancer-associated fibroblasts, and tumor-associated macrophages. CD68+ macrophages isolated from tumorigenic spheroids exhibited profound induction of phenotypic endothelial characteristics. The results are zebrafish tumor xenograft model and by using human patient samples. This multicellular 3D tumor model is a promising tool to study tumor-stroma interaction and cellular plasticity, targeting tumor heterogeneity, and facilitating cancer therapy success against NSCLC.