In recent years, there have been significant strides in the area of cancer research, particularly with the emergence of 3D spheroids as a model system for drug screening and therapeutics development for solid tumour [
1]. Spheroids are three-dimensional (3D) cell culture models that mimic the architecture and microenvironment of solid tumours more closely than traditional two-dimensional (2D) monolayer cultures [
1]. Tumour spheroids are generated by culturing cancer cells in a 3D environment, allowing them to self-assemble into multicellular aggregates. This 3D structure enables the formation of cell–cell interactions, leading to the development of complex tumour-like structures [
2]. Glucose metabolism plays a crucial role in cancer development and progression of cancer. In the tumour microenvironment (TME), including the immune cells such as macrophages, significantly influences metabolic reprogramming in cancer cells [
3]. Glucose is primarily metabolised through glycolysis, leading to the production of pyruvate, which is then converted to lactate rather than being fully oxidised in the mitochondria. Whereas cancer cells exhibit altered glucose metabolism, known as the Warburg effect or aerobic glycolysis, characterised by increased glucose uptake and lactate production, even under oxygen-sufficient conditions [
3]. This metabolic phenotype allows cancer cells to meet their energy demands and support rapid proliferation. Macrophages are key immune cells present in the tumour microenvironment and can be polarised into distinct phenotypes, including pro-inflammatory M1 and anti-inflammatory M2 macrophages [
4]. Macrophages play a dual role in cancer, with M1 macrophages exhibiting anti-tumour properties, while M2 macrophages promote tumour growth and immune evasion [
4]. The metabolic phenotype of macrophages also influences their functions and interactions with cancer cells [
3]. Macrophages exhibit a metabolic plasticity that allows them to adapt to different microenvironments [
5]. Resting macrophages, known as M0 macrophages, primarily rely on oxidative phosphorylation (OXPHOS) for energy production [
3,
6]. Upon activation, M1 macrophages shift towards glycolysis to support their pro-inflammatory functions, while M2 macrophages rely more on OXPHOS to support their immunosuppressive and tissue remodelling functions [
6]. Cancer cell-derived factors, such as cytokines (e.g., interleukin-6, tumour necrosis factor alpha) and chemokines (e.g., CCL2, CCL5), can polarise macrophages towards the M2 phenotype, which promotes a shift towards OXPHOS and supports tumour growth [
7]. which influences the glucose metabolism of macrophages in the TME. Additionally, cancer cells release lactate, which can be taken up by M2 TAMs and utilised as a fuel source through OXPHOS [
8]. The metabolic interplay between cancer cells and macrophages has significant implications in cancer progression. M2 TAMs, fuelled by lactate and oxidative metabolism, promote tumour growth, angiogenesis, and immunosuppression [
8]. The metabolic reprogramming of macrophages towards M2 TAMs phenotype can also influence the tumour microenvironment by altering nutrient availability and immune responses, facilitating tumour immune evasion and metastasis [
3]. TAMs are known to exhibit diverse phenotypes, including pro-tumoral and immunosuppressive characteristics, which can contribute to reduce efficacy of chemotherapeutic drugs which is referred to as TAM-Mediated chemo resistance [
9]. Cells in TME secrete various growth factors, cytokines, and chemokines that promote tumour cell survival and proliferation. TAMs upregulate the expression of drug efflux pumps, such as P-glycoprotein, which actively transport chemotherapeutic agents out of cancer cells [
10]. This efflux mechanism reduces intracellular drug concentrations and limiting their cytotoxic effects. Furthermore, TAMs re-model the TME by secreting extracellular matrix components and promoting angiogenesis which alters TMEs architecture, create physical barriers and hinder drug penetration into the tumour, reducing drug efficacy [
11]. Incorporating macrophages into tumor spheroids to study the interaction between cancer cells and macrophages poses distinct challenges due to the intricate nature of their interactions. Optimising the macrophage-to-cancer cell ratio and culturing conditions is crucial to maintain spheroid integrity and functionality [
12]. In this article we have explored the potential strategy to co-culture macrophages in tumour spheroids to recapitulating key features of solid tumours, including cellular heterogeneity, glucose metabolism, nutrient gradients, ROS emission, and interplay between hypoxia and HSP70 for ECM degradation enzyme upregulation to making a more physiologically relevant spheroid model for drug Screening.