Although there have been many advances in recent years for the treatment of colorectal cancer (CRC), it still remains the third most common cause of cancer-related deaths worldwide. Many patients with late stage CRC display resistance to multiple different therapeutics. An important aspect in developing effective therapeutics for CRC patients is understanding the interactions that take place in the tumor microenvironment (TME), as it has been shown to contribute to drug resistance in vivo. Much research over the past 100 years has focused on 2D monolayer cultures or in vivo studies, however, the efficacy in translating these to the clinic is very low. More recent studies are turning towards developing an effective 3D model of CRC that is clinically relevant, that can recapitulate the TME in vitro and bridge the gap between 2D cultures and in vivo studies, with the aim of reducing the use of animal models in the future. This review summarises the advantages and limitations of different 3D CRC models. It emphasizes how different 3D models may be optimised to study cellular and extracellular interactions that take place in the TME of CRC in an effort to allow the development of more translatable effective treatment options for patients.

Checkpoint blockade immunotherapies have revolutionised cancer treatment in the last decade. Nevertheless, these are only beneficial for a small proportion of cancer patients. Important prognosticators for response to immunotherapy are the mutation burden of tumours as well as the quality and quantity of tumour‐infiltrating immune cells. High‐throughput multiplex immunophenotyping technologies have a central role in deciphering the complexity of anti‐tumour immune responses. Current techniques for the immunophenotyping of solid tumours are held back by the lack of spatial context, limitations in the number of targets that can be visualised simultaneously, and/or cumbersome protocols. We developed a tyramide signal amplification‐free method for the simultaneous detection of seven cellular targets by immunofluorescence. This method overcomes limitations posed by most widespread techniques and provides a unique tool for extensive phenotyping by multispectral fluorescence microscopy. Furthermore, it can be easily implemented as a high‐throughput technology for validation of discovery sets generated by RNA sequencing or mass cytometry and may serve in the future as a complementary diagnostic tool.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. CRC develops in a complex tumour microenvironment (TME) with both mesenchymal stromal cells (MSCs) and immune infiltrate, shown to alter disease progression and treatment response. We hypothesised that an accessible, affordable model of CRC that combines multiple cell types will improve research translation to the clinic and enable the identification of novel therapeutic targets. A viable gelatine-methacrloyl-based hydrogel culture system that incorporates CRC cells with MSCs and a monocyte cell line was developed. Gels were analysed on day 10 by PCR, cytokine array, microscopy and flow cytometry. The addition of stromal cells increased transcription of matrix remodelling proteins FN1 and MMP9, induced release of tumour-promoting immune molecules MIF, Serpin E1, CXCL1, IL-8 and CXCL12 and altered cancer cell expression of immunotherapeutic targets EGFR, CD47 and PD-L1. Treatment with PD153035, an EGFR inhibitor, revealed altered CRC expression of PD-L1 but only in gels lacking MSCs. We established a viable 3D model of CRC that combined cancer cells, MSCs and monocytic cells that can be used to research the role the stroma plays in the TME, identify novel therapeutic targets and improve the transitional efficacy of therapies.

Although substantial advances have been made in cancer immunotherapy, the immunosuppressive tumour microenvironment (TME) continues to pose a significant obstacle to the achievement of sustained clinical responses. Cancer‐associated fibroblasts (CAFs) are increasingly recognised as pivotal regulators in the TME. Their functional adaptability shapes immune responses through extracellular matrix (ECM) remodelling, metabolic reprogramming and the secretion of immunomodulatory factors. This review explores the heterogeneity and plasticity of CAFs, characterising them as dynamic functional states, including myofibroblastic (myCAF), inflammatory (iCAF), antigen‐presenting (apCAF) phenotypes and hypoxia‐induced senescent fibroblasts (hsCAF), which transition fluidly in response to microenvironmental cues and oncogenic signalling. Through a comparative analysis of colorectal cancer (CRC) and multiple myeloma (MM), this review highlights how stromal regulation is adaptive across solid and haematological malignancies. In CRC, CAFs establish dense, collagen‐rich ECM barriers that exclude effector T cells, whereas, in MM, they establish adhesion‐dependent niches within the bone marrow to spatially segregate immune cells from malignant plasma cells. This review also discusses how oncogenic drivers such as KRAS and TP53 ‘educate’ the stromal microenvironment and explores emerging immune evasion mechanisms, including the sialic acid‐Siglec glyco‐immune checkpoint. Current therapeutic approaches are shifting from non‐selective stromal depletion toward normalising phenotypes, blocking secretome factors and disrupting metabolic interactions. The integration of spatial multi‐omics and artificial intelligence provides a framework for mapping these functional landscapes. In conclusion, harnessing CAF heterogeneity in multimodal combination therapies may help shift the tumour microenvironment from an immunosuppressive to a more pro‐immune state. image

The tumour microenvironment (TME) comprises a dynamic network of structural, cellular, and signalling components that interact to influence tumour development. Immune cells play a key role in recognizing and eliminating cancer cells through detection of tumour‐derived membrane and secreted proteins processed in the endoplasmic reticulum (ER). These ER‐matured proteins on target cells act as critical communication signals, coordinating immune responses against tumour cells. However, during tumorigenesis, accumulation of unfolded or misfolded proteins in the ER of the cancer cells triggers the unfolded protein response (UPR), a transcriptional program that alleviates proteotoxic stress by reshaping the cancer cell proteome and secretome, thereby promoting survival. Activation of UPR disrupts intercellular communication and ultimately impairs immune‐mediated tumour clearance. Understanding this crosstalk could inform the development of therapeutics targeting UPR‐driven immune modulation. This review discusses how UPR‐signalling modulates tumour‐immune cell interactions within the TME.