Spatially resolved whole-transcriptome analysis with simultaneous highly multiplexed immune cell epitope detection in multiple cancer tissues

The development and progression of solid tumors such as colorectal cancer (CRC) are known to be affected by the immune system and cell types such as T cells, natural killer (NK) cells, and natural killer T (NKT) cells are emerging as interesting targets for immunotherapy and clinical biomarker research. In addition, CD3+ and CD8+ T cell distribution in tumors has shown positive prognostic value in stage I–III CRC. Recent developments in digital computational pathology support not only classical cell density based tumor characterization, but also a more comprehensive analysis of the spatial cell organization in the tumor immune microenvironment (TiME). Leveraging that methodology in the current study, we tried to address the question of how the distribution of myeloid derived suppressor cells in TiME of primary CRC affects the function and location of cytotoxic T cells. We applied multicolored immunohistochemistry to identify monocytic (CD11b+CD14+) and granulocytic (CD11b+CD15+) myeloid cell populations together with proliferating and non-proliferating cytotoxic T cells (CD8+Ki67+/–). Through automated object detection and image registration using HALO software (IndicaLabs), we applied dedicated spatial statistics to measure the extent of overlap between the areas occupied by myeloid and T cells. With this approach, we observed distinct spatial organizational patterns of immune cells in tumors obtained from 74 treatment-naive CRC patients. Detailed analysis of inter-cell distances and myeloid-T cell spatial overlap combined with integrated gene expression data allowed to stratify patients irrespective of their mismatch repair (MMR) status or consensus molecular subgroups (CMS) classification. In addition, generation of cell distance-derived gene signatures and their mapping to the TCGA data set revealed associations between spatial immune cell distribution in TiME and certain subsets of CD8+ and CD4+ T cells. The presented study sheds a new light on myeloid and T cell interactions in TiME in CRC patients. Our results show that CRC tumors present distinct distribution patterns of not only T effector cells but also tumor resident myeloid cells, thus stressing the necessity of more comprehensive characterization of TiME in order to better predict cancer prognosis. This research emphasizes the importance of a multimodal approach by combining computational pathology with its detailed spatial statistics and gene expression profiling. Finally, our study presents a novel approach to cancer patients’ characterization that can potentially be used to develop new immunotherapy strategies, not based on classical biomarkers related to CRC biology.

Tissue-resident immune cells differ from their corresponding blood cells in many functional aspects. Although the proteome of blood immune cells has been well-investigated, there are almost no data on tissue-resident immune cells. Here, we explored the potential of using MALDI-TOF-MS imaging (MSI) to investigate these cells in colon tissue, which exhibits a strong infiltration of immune cells. MSI identified several proteinaceous markers that colocalized with specific structures of the colon, such as mucosa or muscularis mucosae, in six patients. In addition, we showed that certain m/z values have the same spatial distribution as CD3+ T lymphocytes in the lymphoid follicular structures or as CD206+ macrophages in the lamina propria. For further corroboration, blood lymphocytes and monocytes from 10 healthy volunteers were analyzed by intact cell mass spectrometry (ICMS). Furthermore, we analyzed monocyte-derived macrophages that had been polarized in vitro into proinflammatory M1 and anti-inflammatory M2 phenotypes. The mass spectra differed clearly among all immune cell types. Additionally, it was found that distinct signals from ICMS analysis were identical to the m/z values found in the MSI experiment in lymphoid follicular structures. These data show for the first time that MSI is well-suited to visualize the spatial distribution of immune cells in human colon tissue. We consider MALDI mass spectrometry imaging to be a technique with high potential for use in rapid investigations of tissue-specific features of cells.

Introduction. The interaction between malignant cells (MCs), stromal cells, tumor-associated lymphocytes (TILs), and tumor-associated macrophages (TAMs) is relevant for non-small cell lung carcinoma (NSCLC) progression. The spatial distribution of those cells may affect the prognosis and can be related to genetic intra-tumor heterogeneity (ITH). The aim of this study was to characterize the immunologic ITH and the spatial distribution of immune cells to MCs in primary NSCLC tumors at early stages using multiplex immunofluorescence (mIF) and image analysis approaches. Material and methods. We studied 33 surgically resected NSCLC cases (adenocarcinomas=23; squamous-cell carcinomas=10) with a history of recurrence in a follow-up of at least 60 months (recurrence, N=13; non-recurrence, N=15). Consecutive FFPE tissue sections were stained with two mIF panels (panel 1: cytokeratin (AE1/AE3), PD-L1, PD-1, CD3, CD8, and CD68; panel 2: AE1/AE3, CD3, CD8, granzyme-B, CD45RO, and FOXP3). Three intra-tumor regions (3mm2 each) per case were selected after gridding the whole tumor section. A total of 99 intratumor regions were scanned and analyzed using Vectra Multispectral-Microscope and InForm-software. From each intratumor region, TILs and TAMs densities, as well as the coefficient of variation, were evaluated. The median distance and the G-Cross area under the curve (AUC) for specific radial distances (10µm, 20µm, and 40µm) were obtained between TILs and TAMs phenotypes to MCs. Results. Recurrence was associated with higher MCs density and TAMs/TILs ratio, and lower TIL densities. A high ITH of cytotoxic T-cells (CTLs) PD-L1+ was associated with worse survival. The distance of TAMs PD-L1+ to MCs PD-L1 negative (60µm vs 25µm) or to MCs PD-L1 positive (25µm vs 13µm) was higher in the non-recurrence group than in recurrence group. Close TAMs PD-L1+ to MCs was associated with worst survival. In a radial distance of 10µm, 20µm, and 40µm, a higher infiltration of CTLs PD-1+, was observed in the group of recurrence than non-recurrence group, surrounding MCs PD-L1 negative (AUC 0.49, 3.80, and 20.03; vs AUC 0.01, 0.16, and 1.29, respectively), and MCs PD-L1 positive (AUC 0.60, 4.35, and 19.90; vs AUC 0.01, 0.20, and 2.20, respectively). A high infiltration of CTLs PD-1+ surrounding MCs, with or without expression of PD-L1, was associated with worse survival. All the differences were statistically significant (P<0.05). Conclusion. Close spatial proximity of antigen-experienced CTLs and TAMs PD-L1+ to MCs are associated with recurrence and poor survival in early stages of NSCLC. We determined that ITH of immune cell densities is associated with recurrence of surgically resected NSCLC. Tumor-immune cell spatial modeling offers a deep understanding of tumor microenvironment that impacts on clinical outcomes. Supported by CPRITRP160668 and UT Lung SPORE grants Citation Format: Alejandro Francisco-Cruz, Edwin R. Parra, Santhoshi N. Krishnan, Souptik Barua, Mei Jiang, Junya Fujimoto, Christine B. Peterson, Priyam Das, Chi-Wan Chow, Jaime Rodriguez-Canales, Carmen Behrens, Neda Kalhor, Annikka Weissferdt, John Heymach, Stephen Swisher, Boris Sepesi, Arvind Rao, J. Jack Lee, Cesar Moran, Andrew Futreal, Jianjun Zhang, Ignacio I. Wistuba. Impact of the spatial analysis of tumor-associated lymphocytes and tumor-associated macrophages on recurrence at early stage of non-small cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1180.

In vitro, MTCQ1, MOCL1, and MOCL2 murine HNSCC cell responses to 2Gyx6 X-ray EBRT were assessed in metabolic, clonogenic and γH2AX assays. Ex vivo, syngeneic tumors in C57BL/6 mice were stained for haematoxylin and eosin (H&E) and immune cell infiltration. Combination therapeutic in vivo studies using MTCQ1 tumors treated with 2Gyx6 or 8Gyx1 EBRT with sequential and/or concurrent dosing with anti-PD-L1 were also performed. MTCQ1 cells exhibited the most marked responses to EBRT in vitro. H&E analysis revealed highest cellular density and most disperse extracellular matrix in MTCQ1 tumors with infiltration of CD8a+T cells in tumor centres and macrophages predominantly peripheral. The 2Gyx6 EBRT regimen slowed tumor progression; average tumor volumes were 129.2±49.0mm3 on day 10, compared to 234.1±130.7mm3 in the CT-only control (P=0.039). However, there was also a reduced CD8a+T cell infiltration on day 3 post complete treatment, with 0.19±0.17% CD8a+T cell area compared to 0.91±0.31% in the CT-only control. Combining EBRT with anti-PD-L1 (delivered either concurrently or sequentially), resulted in greater median survival compared to the CT-only control (33 and 32days versus 28days, respectively). Similarly, statistically insignificant improved survival with 8Gyx1 EBRT regimen combined with concurrent anti-PD-L1 was observed. These results reveal spatial distribution of immune cells in the tumor microenvironment and underscore the role of EBRT regimens in modulating immune cell dynamics. This highlights the importance of optimising radiation protocols to inform combination therapy designs in preclinical models.

Long-lasting immunity depends on generation of antibody forming cells in germinal centers (GCs). Conventional methods such as immunohistology and intravital live imaging have been used extensively to investigate the location of cellular assemblies within tissues as well as their dynamic motility and cellular interactions. Two photon laser scanning microscopy (TPLSM) intravital imaging allows scanning of large areas within tissues and reveals multiple immune cell niches. Nonetheless, this type of imaging is limited by the depth of penetration and cannot capture effectively all of the GC niches within lymphoid organs. Here we describe a method to visualize antigen-specific T and B cells in multiple microanatomical locations and niches at the level of a whole organ. This large-scale imaging approach can greatly increase our understanding of the spatial distribution of immune cells and help obtain detailed 3D maps of their locations and quantities.

Assessing the Immune Tumour Microenvironment (iTME) Using Multiplex Immunoflourescence Histochemistry (mIHC) Demonstrates Close Proximity of Cytotoxic T-Cells to Plasma Cells (PC) in Patients with Newly Diagnosed Multiple Myeloma (NDMM)

Elevated expression of Inducible Nitric oxide Synthase-2 (NOS2) occurs during acute and chronic inflammation and wound healing processes however, it is also a predictor of bad prognosis in aggressive ER-negative tumors. Overexpression of NOS2 correlates with other prognostic biomarkers making it a unique multifunctional oncoprotein. Cyclooxygenase-2 (COX2) is another enzyme associated with cancer inflammation and is overexpressed in >50% of breast cancers. In our previous studies, we have shown that NOS2/COX2 co-expression is a strong predictor of poor clinical outcome and promotes CD8+ T cell exclusion from tumor epithelia in Triple-negative breast cancers (TNBC). This observation was further explored using multiplexing proteomics technologies. Imaging Mass Cytometry (IMC) has revolutionized tumor microenvironment research by allowing the interrogation of over 35 protein markers simultaneously for the elucidation of the spatial distribution of immune and tumor cells at sub-cellular resolution. Herein, we used multiplex capability IMC to explore the interactions of immune and tumor cell counterparts in ER-negative and TNBC tumors. We employed Formalin-fixed paraffin-embedded (FFPE) tissue from 26 ER-negative and TNBC samples from a clinical cohort of African American breast cancer patients in the Baltimore MD area. We developed a panel of 15 protein metal-labeled antibody markers including antibodies targeting NOS2, COX2, T cells (CD45, CD3, CD4, CD8,) endothelium (CD31), monocytes (CD14), macrophages (CD68), basal markers (α-SMA), cancer stem cells (CD44v6), cancer cells (Pan-CK, B7H4, Cadherin), and serine proteases (granzyme B) secreted by T cells or Natural Killer (NK) cells. Single section slides of 4 µm size were stained with the cocktail of metal-labeled antibodies. Multiple regions of interest (ROIs) representing the tumor and immune cells compartments and tumor-facing stromal cell areas were ablated with the Hyperion Imaging system. The data analysis was performed by the Bodenmiller lab pipeline and VisioPharm software packages. Our preliminary data suggest that patient tumors with high or low NOS2/COX2 expression showed variable levels of CD68 positive macrophage population and T cells. We are currently analyzing the remaining data to understand the cell phenotypes associated with the immunosuppressive and immune-rich clinical samples and performing neighborhood analysis of tumor cells to immune cells and vessels. Citation Format: Milind Pore, Lisa Ridnour, Paul Mallory, Ashley Cardamone, Stephen Lockett, William Bocik, David Wink. Multiplex proteomics profiling with the imaging mass cytometry (IMC) identifies immune cell phenotypes associated with high NOS2/COX2 expression in ER- negative and TNBC patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3588.

BackgroundThe tumor microenvironment (TME) is composed of highly heterogeneous cell types that dynamically interact with each other. To more precisely distinguish and characterize cellular diversity and disease states within tissues,...

<p>Supplementary Figure 6. iTIL cell distribution according to NAC response in TNBC F-H) Staining of selective markers to detect the presence of CD20+ B lymphocytes (F), CD4+ T helper lymphocytes (G) and ERG+ endothelial cells (H) comparing pCR and pNR patients that had a similar percentage of sTILs at diagnosis. In each slide, an intra-epithelial area of the lesion is highlighted with higher magnification to show iTILs (left 5X magnification with scale bar 200µm; right 20X magnification with scale bar 50µm).</p>

Combining physical simulation and statistical modeling methods to study the spatial distribution of immune cells in lung cancer microenvironment

Immune response plays a significant role in the progression of neurodegenerative diseases (NDs) such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and multiple sclerosis (MS). It is paramount to quantify the immune and structural spatial microenvironment of ND brain using research approaches that resolve tissue at a single-cell level. Imaging Mass Cytometry™ (IMC™) is a spatial biology technique that provides quantitative phenotypic evaluation of 40-plus markers. We report the application of two new IMC imaging modes with 40-marker panels for ND research. Tissue mode (TM) was applied to perform a scan of the entire tissue followed by pixel-based analysis (PBA) to uncover spatial distribution of relevant markers. Preview mode (PM) was applied to sample the tissue at low resolution, improving informed selection of areas for high-resolution cell mode (CM) imaging on the same slide. Using TM with PBA, we quantified the spatial distribution of immune cells in tissue compartments of ND tissues. PM and CM highlighted MS lesions devoid of myelinization and a network of α-synuclein in PD tissue. Single-cell analysis identified heterogeneity of amyloid precursor protein aggregates in AD that encompassed total and phosphorylated species of tau and phosphorylated α-synuclein. Overall, whole slide imaging techniques using IMC provide quantitative immunological and structural insights into the spatial biology of ND. For Research Use Only. Not for use in diagnostic procedures.

BackgroundAberrations in homologous recombination repair (HRR) genes are emerging as important biomarkers for personalized treatment in prostate cancer (PCa). HRR deficiency (HRD) could affect the tumor immune microenvironment (TIME), potentially...

<p>Supplementary Figure 6. iTIL cell distribution according to NAC response in TNBC F-H) Staining of selective markers to detect the presence of CD20+ B lymphocytes (F), CD4+ T helper lymphocytes (G) and ERG+ endothelial cells (H) comparing pCR and pNR patients that had a similar percentage of sTILs at diagnosis. In each slide, an intra-epithelial area of the lesion is highlighted with higher magnification to show iTILs (left 5X magnification with scale bar 200µm; right 20X magnification with scale bar 50µm).</p>

<p>Supplementary Figure 5. Gene expression and RCB score</p>

Gastrointestinal stromal tumors (GIST) are the most prevalent mesenchymal tumor entity of the gastrointestinal tract and serve as a key example for targeted therapies with tyrosine kinase inhibitors (TKI) such as imatinib, in tumors with the most frequent mutations in PDGFRA or c-Kit. Although TKIs have significantly improved the overall survival rates of GIST patients, there are currently no treatment options for imatinib-resistant tumors and tumors without identifiable driver mutations. So far, current sarcoma research mostly relies on expensive mouse models or 2D cell culture that do not integrate the tumor microenvironment (TME). Therefore, clinically relevant, patient-derived 3D GIST models that include cells of the TME are urgently needed. In this study, we tested the feasibility of precision-cut tumor slices (PCTS) as a preclinical model for personalized GIST research. PCTS are patient-derived 3D tissue slices that enable the study of interactions between tumor cells and cells of the surrounding TME. For this, primary GIST tumors were cut into 250µm thick slices and cultured for up to two weeks with or without standard TKI treatment. Tumor morphology and cell composition was analyzed via immunohistochemical (IHC) and multiplex immunofluorescence (mIF) staining, which enabled the simultaneous detection of up to six different markers on one FFPE tissue section. The downstream analysis included a machine-learning based workflow that allowed the comparison of biomarker expression and spatial immune cell distribution in the PCTS stromal and tumor areas before and after treatment. Comparison between primary tumor and PCTS revealed a preserved tumor morphology and heterogeneous cell composition throughout culture. In addition, serum levels of various cytokines were assessed with LEGENDPlex assays and correlated with the PCTS cell composition. The in vitro response of PCTS to TKI-based therapies was analyzed via IHC staining and ATP-based viability assays. PCTS from tumors with PDGFRA mutations were expected to be imatinib-resistant, while PCTS from tumors with c-KIT mutations were considered imatinib-sensitive. Sequencing analyses determined the PDGFRA or c-KIT mutational status showing that all patients harbored either a PDGFRA or a c-KIT mutation. PCTS treatment response was compared to the mutational status and if available to clinical follow-up data. Of all PCTS that responded to imatinib therapy, two cases matched the anticipated clinical outcome. PCTS provide a clinically relevant new model system for GIST to explore the development of therapy response to TKI-based treatment and to investigate novel GIST treatments such as immunotherapies and combination treatments. The work is supported by the Robert Bosch Stiftung, Stuttgart, Germany Citation Format: Dina Mönch, Julia Thiel, Meng Dong, Annika Maaß, Annette M. Staiger, Katrin Kurz, German Ott, Philipp Renner, Tobias Leibold, Thomas Mürdter, Matthias Schwab, Marc-Hendrik Dahlke, Jana Koch. Evaluation of individual drug response to tyrosine kinase inhibitor treatment in precision-cut tumor slices of gastrointestinal stroma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6845.