Tumor microenvironment (TME) multiplex immunophenotyping analyses

The use of preclinical orthotopic tumors to better recapitulate the tumor microenvironment (TME)

Complex interactions between the cellular and structural components of the tumor microenvironment (TME) composed of tumor cells, fibroblasts, endothelial cells, immune cells, extracellular matrix components etc., play a significant role in development and metastasis of tumors. Accordingly, the continuous interactions between tumor cells and the TME may also impact your novel drug’s efficacy or even lead to drug resistance. However, in many instances, subcutaneously grown tumor models do not fully recapitulate the TME. Therefore, orthotopically injected tumor cells arise as a better way to model some types of cancer, such as colorectal cancer (Figure 1). More advanced studies may also benefit from studies evaluating efficacy in patient-derived xenografts and patient-derived organoids in a co-culture setting to determine TME interactions in vitro.  

Still, most preclinical studies focus on non-spatial analyses, missing the opportunity to fully evaluate the interactions between TME, tumors and how they impact drug efficacy and pharmacodynamics. In this case study using colon adenocarcinoma tumor model (MC38), we show how multiplex immunofluorescence analyses can help you uncover preserved spatial TME and immune cell interactions, which in turn can help predict your immunotherapy’s response and evaluation.  

FIGURE 1. The colorectal adenocarcinoma (MC38) model is offered at InnoSer both as a subcutaneous (MC38) and orthotopic model (MC38-luc) model. Disease progression is assessed via observation of clinical signs, body weight, and tumour volume measurements (callipers or bioluminescence imaging in case of orthotopic injections). This model shows efficient growth kinetics and response to anti-PD-1 therapy. The left figure shows tumour volume measured by callipers (subcutaneous model), mean ± SEM, **P<0.01, ***P<0.001. The right figure shows tumour progression in a subset of two mice in the orthotopic MC38 model.  

Multiplexed IF analyses provide deeper insight into the spatial (preserved) tumor microenvironment (TME) immune cell interaction and immunotherapy response prediction and evaluation 

By performing your studies together with experts at InnoSer, you can gain access to both non-spatial immunophenotyping and TME analyses via flow cytometry and/or meso-scale discovery (MSD) as well as spatial analyses via classical and advanced histology methods. Spatial TME profiling via multiplex IF labelling and analyses arises as a highly suitable method to help you better understand the effects and/or interactions of your novel therapy with the TME. In Figures 2-4 we show an example of multiplex IF analyses on tumor samples obtained from MC38 colorectal adenocarcinoma samples (subcutaneously implanted) from untreated (vehicle) and anti-PD-1 treated mice.  

Following studies performed using in vivo models, InnoSer can support you to better understand the dynamic crosstalk between tumor cells and the TME, offering multiplex immunofluorescence staining analyses consisting of multiple panels (T cells, B cells, plasma cells, myeloid cells, neutrophils, tumor compartment cells etc.,). Compared to traditional cell profiling methods like flow cytometry, multiplex IF allows you to study complex cell-cell interactions, cellular infiltration, the 3D immune profile, signaling proteins etc., in preserved tumor tissue. At InnoSer, TME analyses can be complemented with a wide range of in vitro assays (co-culture assays, angiogenesis etc.,).  

FIGURE 2. Example of an application of immunofluorescence multiplex analyses. (A) Spatial TME analyses help localize cells in different areas. This tumor sample staining obtained from MC38 adenocarcinoma mouse model (implanted subcutaneously) showcases a multiplex panel of antibodies to detect B-cells (beige), colorectal cancer cells (red), epithelium (purple), M1 macrophages (light blue), M2 macrophages (dark blue), neutrophils (green), T-cells (yellow), cytotoxic T-cells (light orange) and T-helper cells (gray). (B) Highlighted areas show preserved spatial analyses showcasing tumoral area (red), peritumoral (yellow) and non-tumoral area (green). Besides defining areas in the tissue, spatial TME analyses are useful to study cellular phenotypes in the tissue context as well as studying cell-cell interactions using complex bioinformatic analyses (i.e., neighbourhood and nearest neighbour analysis).  

FIGURE 3. Example of quantitative cytometric profiling with spatial resolution based on multiplex immunofluorescence imaging. (A) Representative multiplex IF staining from MC38 colorectal carcinoma mouse model (implanted subcutaneously) tumor sample, stained for B-cells, CRC, epithelium, macrophages (M1 and M2), neutrophils, T-cells, cytotoxic T-cells and T-helper cells. (B) Segmentation of the areas into tumoral (red), peritumoral (yellow) and non-tumoral areas (green). (C) Percentage of different cell types in each of the areas defined within the obtained tumor tissue sample. Multiplex IF staining visualisation software allow to view and analyse different markers at different times, here shown are sub-stainings of (D) CD3, CD19, CD11c (E) CD206, F480, panCK and (F) CD4, CD8, p53, MPO.  

FIGURE 4. Example of an application of cytometric profiling with spatial resolution based on multiplex immunofluorescence imaging to obtain quantitative results. Quantitative analyses reveal differences in immune cell infiltration between vehicle–treated and PD-1 treated MC38 colorectal carcinoma tumours (implanted subcutaneously).