Ex-vivo Clonally Expanded B Lymphocytes Infiltrating Colorectal Carcinoma Are of Mature Immunophenotype and Produce Functional IgG

Background: Tumor infiltrating B cells (TiBc) have not yet been investigated in detail. This may at least in part be due to technical difficulties. Here we describe a straightforward and reproducible method to isolate and culture TiBc from primary colorectal carcinomas (CRC). Methods/Results: TiBc cultures were generated by Epstein-Barr virus (EBV) immortalization. With this method, monoclonal TiBc cultures were obtained for 14/19 CRCs. As assessed by flow cytometry and ELISA, TiBc showed an activated immunophenotype (CD23 +, CD80 +) and produced immunoglobulin (Ig; IgG secretion in 55 % of the cultures). In functional in vitro analysis, most of the IgGs specifically bound to allogeneic CRC target cells. These data suggest that TiBc are antigenexperienced and thus may exhibit functionality in situ. Additionally, mini-cultures generated from 12 further CRCs revealed TiBc outgrowth exclusively in the presence of EBV. Conclusion: In summary, this simple method provides a cellular tool and our data set the stage for analysing the bivalent role of TiBc; being antigen-presenting cells on the one hand and tumor-specific antibody producers on the other. Additionally, the generation of long-term TiBc cultures and their monoclonal Ig may serve to identify novel tumor-specifi

Cryopreservation of human colorectal carcinomas prior to xenografting

<p>Abstract</p> <p>Background</p> <p>Molecular heterogeneity of colorectal carcinoma (CRC) is well recognized, forming the rationale for molecular tests required before administration of some of the novel targeted therapies that now are rapidly entering the clinics. For clinical research at least, but possibly even for future individualized tumor treatment on a routine basis, propagation of patients' CRC tissue may be highly desirable for detailed molecular, biochemical or functional analyses. However, complex logistics requiring close liaison between surgery, pathology, laboratory researchers and animal care facilities are a major drawback in this. We here describe and evaluate a very simple cryopreservation procedure for colorectal carcinoma tissue prior to xenografting that will considerably reduce this logistic complexity.</p> <p>Methods</p> <p>Fourty-eight CRC collected ad hoc were xenografted subcutaneously into immunodeficient mice either fresh from surgery (N = 23) or after cryopreservation (N = 31; up to 643 days).</p> <p>Results</p> <p>Take rates after cryopreservation were satisfactory (71%) though somewhat lower than with tumor tissues fresh from surgery (74%), but this difference was not statistically significant. Re-transplantation of cryopreserved established xenografts (N = 11) was always successful. Of note, in this series, all of the major molecular types of CRC were xenografted successfully, even after cryopreservation.</p> <p>Conclusions</p> <p>Our procedure facilitates collection, long-time storage and propagation of clinical CRC specimens (even from different centres) for (pre)clinical studies of novel therapies or for basic research.</p

Agent-Based Behavioral Modeling of Human Associative Learning in a Complex Approach-Avoidance Conflict Task

This repository contains the code, data, and figures of the correspondent manuscript

Establishment, characterization and chemosensitivity of three mismatch repair deficient cell lines from sporadic and inherited colorectal carcinomas.

BACKGROUND: Colorectal cancer (CRC) represents a morphologic and molecular heterogenic disease. This heterogeneity substantially impairs drug effectiveness and prognosis. The subtype of mismatch repair deficient (MMR-D) CRCs, accounting for about 15% of all cases, shows particular differential responses up to resistance towards currently approved cytostatic drugs. Pre-clinical in vitro models representing molecular features of MMR-D tumors are thus mandatory for identifying biomarkers that finally help to predict responses towards new cytostatic drugs. Here, we describe the successful establishment and characterization of three patient-derived MMR-D cell lines (HROC24, HROC87, and HROC113) along with their corresponding xenografts. METHODOLOGY: MMR-D cell lines (HROC24, HROC87, and HROC113) were established from a total of ten clinicopathological well-defined MMR-D cases (120 CRC cases in total). Cells were comprehensively characterized by phenotype, morphology, growth kinetics, invasiveness, and molecular profile. Additionally, response to clinically relevant chemotherapeutics was examined in vitro and in vivo. PRINCIPAL FINDINGS: Two MMR-D lines showing CIMP-H derived from sporadic CRC (HROC24: K-ras(wt), B-raf(mut), HROC87: K-ras(wt), B-raf(mut)), whereas the HROC113 cell line (K-ras(mut), B-raf(wt)) was HNPCC-associated. A diploid DNA-status could be verified by flow cytometry and SNP Array analysis. All cell lines were characterized as epithelial (EpCAM(+)) tumor cells, showing surface tumor marker expression (CEACAM(+)). MHC-class II was inducible by Interferon-γ stimulation. Growth kinetics as well as invasive potential was quite heterogeneous between individual lines. Besides, MMR-D cell lines exhibited distinct responsiveness towards chemotherapeutics, even when comparing in vitro and in vivo sensitivity. CONCLUSIONS: These newly established and well-characterized, low-passage MMR-D cell lines provide a useful tool for future investigations on the biological characteristics of MMR-D CRCs, both of sporadic and hereditary origin. Additionally, matched patient-derived immune cells allow for comparative genetic studies

Representative immunofluorescence images of tumor cells showing positive binding of both membranous as well as intracellular target structures by TiBc-derived IgGs.

<p>(A) HCT116 and (B) HROC46 tumor cells were stained with TiBc-derived IgGs and Alexa 546-conjugated anti-human IgG. Cell nuclei were counterstained with DAPI. Therapeutic antibodies Cetuximab and Rituximab served as positive and negative control, respectively. Original magnification ×40.</p

Tumor-infiltrating lymphocytes in a primary colorectal carcinoma (HROC60) highlighted by immunohistochemistry.

<p>(A, B) TiBc are observed to concentrate in follicular aggregates at the invasive margin. (C, D) Diffuse T cell infiltration within tumor stroma and to a minor extend within neoplastic glands given by positive staining for CD3. (E) TiBc in follicular aggregates (arrows) reside in a mesh of follicular dendritic cells (inset). Original magnification (A, C, E) 4× and (B, D) 20×.</p

Data of colorectal carcinomas used for generation of TiBc bulk cultures, flow cytometry of CD19⁺CD20⁺ B cells from primary tumors and overall results of outcomes.

<p>NA – not analyzed;</p>†<p>- no long-term culture established;</p><p>ND – not yet determined.</p

<i>In vivo</i> tumorigeneicity & response to selected chemotherapeutics.

<p>NMRI nu/nu mice either received s.c. injections of established MMR-D cell lines (5 Mio. cells/mouse; A–C) or were s.c grafted with HROC29 and HROC71 tumor fragments (D, E). For analyzing <i>in vivo</i> drug response, mice with established (A) HROC24, (B) HROC87 or (C) HROC113 tumors were treated with irinotecan, paclitaxel or gemcitabine (i.p.; 20 mg/kg bw each, n = 6−7 mice per group). HROC71 and HROC29 xenografts were given gemcitabine (i.p.; 20 mg/kg bw, n = 5−6 mice per group). Therapeutic regimens consisted of six injections in total, applied twice a week. Control animals received equivalent volumes of saline (n = 6−7). Values of are given as mean ± SEM. *p<0.05 vs. control, <i>U</i>-Test; ^#p<0.05 vs. control, <i>t</i>-test.</p