Macrophage infiltration in 3D cancer spheroids to recapitulate the TME and unveil interactions within cancer cells and macrophages to modulate chemotherapeutic drug efficacy.

BACKGROUND: Recapitulating the tumor microenvironment (TME) in vitro remains a major hurdle in cancer research. In recent years, there have been significant strides in this area, particularly with the emergence of 3D spheroids as a model system for drug screening and therapeutics development for solid tumors. However, incorporating macrophages into these spheroid cultures poses specific challenges due to the intricate interactions between macrophages and cancer cells. METHODS: To address this issue, in this study, we established a reproducible healthy multicellular 3D spheroid culture with macrophage infiltrates in order to mimic the TME and modulate the drug's efficacy on cancer cells in the presence of macrophages. A 3D spheroid was established using the human cancer cell line CAL33 and THP1 cell derived M0 macrophages were used as a source of macrophages. Cellular parameters including tumour metabolism, health, and mitochondrial mass were analysed in order to establish ideal conditions. To modulate the interaction of cancer cells with macrophage the ROS, NO, and H2O2 levels, in addition to M1 and M2 macrophage phenotypic markers, were analyzed. To understand the crosstalk between cancer cells and macrophages for ECM degradation, HSP70, HIF1α and cysteine proteases were examined in spheroids using western blotting and qPCR. RESULTS: The spheroids with macrophage infiltrates exhibited key features of solid tumors, including cellular heterogeneity, metabolic changes, nutrient gradients, ROS emission, and the interplay between HIF1α and HSP70 for upregulation of ECM degradading enzymes. Our results demonstrate that tumor cells exhibit a metabolic shift in the presence of macrophages. Additionally, we have observed a shift in the polarity of M0 macrophages towards tumor-associated macrophages (TAMs) in response to cancer cells in spheroids. Results also demonstrate the involvement of macrophages in regulating HIF-1α, HSP70, and ECM degradation cysteine proteases enzymes. CONCLUSIONS: This study has significant implications for cancer therapy as it sheds light on the intricate interaction between tumor cells and their surrounding macrophages. Additionally, our 3D spheroid model can aid in drug screening and enhance the predictive accuracy of preclinical studies. The strength of our study lies in the comprehensive characterization of the multicellular 3D spheroid model, which closely mimics the TME.

Progressive growth of a murine T cell lymphoma alters population kinetics and cell viability of macrophages in a tumor-bearing host.

Tumor progression induces infiltration of immune cell populations at the site of tumor growth. Infiltrated leukocyte population including monocyte and macrophages interacts with tumor cells and tumor microenvironment and results in the suppression of macrophage functions. Impaired functions of macrophages result in the suppression/inhibition of cell-mediated immunity leading to inefficient antitumor immune responses. Impaired macrophage population invariably helps in immune selection of tumor leading to uninterrupted growth and progression in the host. Murine T cell lymphoma designated as Dalton's lymphoma is highly immunosuppressive and invasive tumor of T cell origin, which completely paralyzes the host's immune system resulting in a very short life span of the host. Progressive growth of Dalton's lymphoma (DL) cells has been known to inhibit the release of inflammatory cytokines and effector mediator molecules. In this study, we demonstrate that intraperitoneal transplant of DL cells in normal healthy host induces a rapid increase in macrophage cell population during early stage of tumor progression and progressive decrease in tumor-associated macrophage population and reduced survival of macrophages in advance stage of tumor burden.

A benzophenanthridine alkaloid, chelerythrine induces apoptosis in vitro in a Dalton's lymphoma.

PURPOSE: The aim of this study was to investigate the effect of chelerythrine on DL cell apoptosis in an in vitro experimental setup. MATERIALS AND METHODS: For tumor model, spontaneous occurring T-cell lymphoma designated as Dalton's lymphoma (DL) was selected. Double staining, transmission electron microscope (TEM), fluorescence microscopy, Western blotting, Reverse Transcriptase-Polymerase Chain Reaction, and DNA fragmentation assay were used to detect heat shock factor 1 (HSF1) and hsp70 expression and PKC phosphorylation, and apoptotic characteristic of DL cells. RESULTS: Chelerythrine exposure resulted in significant morphological alteration comparable to that of apoptosis. Furthermore, it was confirmed by fluorescence microscopy, TEM analysis, and DNA fragmentation assay that 10 µg/mL of chelerythrine is capable of inducing apoptosis in DL cells. The suppression in HSF1 expression and subsequent inhibition of hsp70 expression in chelerythrine-treated DL cells suggest that chelerythrine induces apoptosis in DL cells by inhibiting the expression of these cytoprotective proteins. CONCLUSION: Chelerythrine is capable of inducing apoptosis DL cells in vitro and therefore, it could be useful in combating tumor growth and progression.