[¹⁸F]FEPPA: Improved Automated Radiosynthesis, Binding Affinity, and Preliminary in Vitro Evaluation in Colorectal Cancer

The overexpression of the translocator protein (TSPO) has been amply reported for a variety of conditions, including neurodegenerative disorders, heart failure, and cancer. Thus, TSPO has been proposed as an excellent imaging biomarker, allowing, in this manner, to obtain an accurate diagnosis and to follow disease progression and therapy response. Accordingly, several radioligands have been developed to accomplish this purpose. In this work, we selected [¹⁸F]­FEPPA, as one of the clinical established tracers, and assessed its in vitro performance in colorectal cancer. Moreover, we setup an improved radiosynthesis method and assessed the in vitro binding affinity of the nonradioactive ligand toward the human TSPO. Our results show an excellent to moderate affinity, in the subnanomolar and nanomolar range, as well as the suitability of [¹⁸F]­FEPPA as an imaging agent for the TSPO in colorectal cancer

In Situ Characterization of Tissue-Resident Immune Cells by MALDI Mass Spectrometry Imaging

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 <i>m</i>/<i>z</i> 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 <i>m</i>/<i>z</i> 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

Intermediate Monocytes but Not TIE2-Expressing Monocytes Are a Sensitive Diagnostic Indicator for Colorectal Cancer

<div><p>We have conducted the first study to determine the diagnostic potential of the CD14++CD16+ intermediate monocytes as compared to the pro-angiogenic subset of CD14++CD16+TIE2+ TIE2-expressing monocytes (TEMs) in cancer. These monocyte populations were investigated by flow cytometry in healthy volunteers (N = 32) and in colorectal carcinoma patients with localized (N = 24) or metastatic (N = 37) disease. We further determined blood levels of cytokines associated with monocyte regulation. The results revealed the intermediate monocyte subset to be significantly elevated in colorectal cancer patients and to show the highest frequencies in localized disease. Multivariate regression analysis identified intermediate monocytes as a significant independent variable in cancer prediction. With a cut-off value at 0.37% (intermediate monocytes of total leukocytes) the diagnostic sensitivity and specificity ranged at 69% and 81%, respectively. In contrast, TEM levels were elevated in localized cancer but did not differ significantly between groups and none of the cytokines correlated with monocyte subpopulations. Of interest, <em>in vitro</em> analyses supported the observation that intermediate monocytes were more potently induced by primary as opposed to metastatic cancer cells which may relate to the immunosuppressive milieu established in the advanced stage of metastatic disease. In conclusion, intermediate monocytes as compared to TIE2-expressing monocytes are a more sensitive diagnostic indicator of colorectal cancer.</p> </div

CD16 expression is increased on monocytes of cancer patients.

<p>The expression level of CD16 and TIE2 on monocyte subsets was determined for healthy individuals and patients with localized or metastatic colorectal cancer. Mean fluorescence intensity (MFI) of CD16-PC5 staining was evaluated for intermediate CD14++CD16+ monocytes (A), and CD14++CD16+TIE2+ TEMs (B). TIE2 expression levels are reflected by MFI of TIE2-PE antibody bound to TEMs (C).</p

Univariate and multivariate analysis by binary logistic regression to calculate significance levels and odds ratios (including 95% confidence intervals) for cancer prediction by the investigated variables.

<p>Univariate and multivariate analysis by binary logistic regression to calculate significance levels and odds ratios (including 95% confidence intervals) for cancer prediction by the investigated variables.</p

Blood levels of intermediate monocytes are a diagnostic indicator of CRC.

<p>The balance in diagnostic sensitivity and specificity of intermediate monocyte levels was evaluated by ROC chart and the area under the curve.</p

Demographic and clinical characteristics of study participants.

<p>N: number of individuals; N.A.: not applicable; N.D.: not determined; UICC: Union for International Cancer Control staging.</p>*<p>Patient with localized CRC was diagnosed based on biopsy material and computed tomography scan. Stage of disease was not determined, as patient did not undergo surgery.</p

B Cells and Ectopic Follicular Structures: Novel Players in Anti-Tumor Programming with Prognostic Power for Patients with Metastatic Colorectal Cancer

<div><p>Remarkably limited information is available about biological mechanisms that determine the disease entity of metastatic colorectal cancer in the liver (CRCLM) with no good clinical parameters to estimate prognosis. For the last few years, understanding the relationship between tumor characteristics and local immune response has gained increasing attention. Given the multifaceted roles of B-cell-driven responses, we aimed to elucidate the immunological imprint of B lymphocytes at the metastatic site, the interrelation with macrophages, and their prognostic relevance. Here we present novel algorithm allowing to assess a link between the local patient-specific immunological capacity and clinical outcome. The microscopy-based imaging platform was used for automated scanning of large-scale tissue sections and subsequent qualitative and quantitative analyses of immune cell subtypes using lineage markers and single-cell recognition strategy. Results indicate massive infiltration of CD45-positive leukocytes confined to the metastatic border. We report for the first time the accumulation of CD20-positive B lymphocytes at the tumor – liver interface comprising the major population within the large CD45-positive aggregates. Strikingly, functionally active, activation-induced cytidine deaminase (AID)-positive ectopic lymphoid structures were found to be assembled within the metastatic margin. Furthermore, the CD20-based data set revealed a strong prognostic power: patients with high CD20 content and/or ectopic follicles had significantly lower risk for disease recurrence as revealed by univariate analysis (p<0.001 for both) and in models adjusted for clinicopathological variables (p<0.001 and p = 0.01, respectively), and showed prolonged overall survival. In contrast, CD68 staining-derived data set did not show an association with clinical outcome. Taken together, we nominate the magnitude of B lymphocytes, including those organized in ectopic follicles, as novel prognostic marker which is superior to clinicopathological parameters. Findings emphasize anti-tumoral role of B cell-driven mechanism(s) and thus indicate a new way of thinking about potential treatment strategies for CRCLM patients.</p></div

Kaplan-Meier plots showing the lung-metastasis free survival, recurrence free survival and overall survival after metastasectomy dependent on stromal Hsp27 (A), stromal α-SMA (B) and tumor Hsp27 (C) scoring.

<p>Kaplan-Meier plots showing the lung-metastasis free survival, recurrence free survival and overall survival after metastasectomy dependent on stromal Hsp27 (A), stromal α-SMA (B) and tumor Hsp27 (C) scoring.</p

The degree of Hsp27 expression score in the tumor stroma correlated significantly with the expression of stromal α-SMA (A).

<p>CD31-positive microvessels surrounded by tumor stroma next to tumor cells (asterisk) (B). MVD was significantly increased in primary tumors and metastases with strong stromal Hsp27 expression (C). Immunofluorescence showed a co-expression of stromal Hsp27 and α-SMA especially in PM (400x magnification) (D).</p