R. Keijzer

A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA.

Apoptosis (programmed cell death) can be difficult to detect in routine histological sections. Since extensive DNA fragmentation is an important characteristic of this process, visualization of DNA breaks could greatly facilitate the identification of apoptotic cells. We describe a new staining method for formalin-fixed, paraffin-embedded tissue sections that involves an in situ end-labeling (ISEL) procedure. After protease treatment to permeate the tissue sections, biotinylated nucleotides are in situ incorporated into DNA breaks by polymerase and subsequently stained with DAB via peroxidase-conjugated avidin. Staining of cells with the morphological characteristics of apoptosis was demonstrated in tissues known to exhibit programmed cell death, i.e., prostate and uterus after castration, tumors, lymph node follicles, and embryos. Apoptotic cells could be discriminated morphologically from areas of labeled necrotic cells, in which DNA degradation also occurs. Because apoptosis is relatively easily recognized in H&E-stained sections of involuting prostates of castrated rats, we used this model system to validate the ISEL method for the quantification of apoptotic cells. A high correlation was found between the fractions of ISEL-labeled cells and the fractions of apoptotic cells that were morphologically determined in adjacent sections. We conclude that ISEL is a useful technique for quantification of apoptosis in paraffin sections, especially for those tissues in which morphological determination is difficult. Furthermore, this new staining method enables the use of automated image cytometry for evaluating apoptosis.

Novel intraoperative near-infrared fluorescence camera system for optical image-guided cancer surgery.

Current methods of intraoperative tumor margin detection using palpation and visual inspection frequently result in incomplete resections, which is an important problem in surgical oncology. Therefore, real-time visualization of cancer cells is needed to increase the number of patients with a complete tumor resection. For this purpose, near-infrared fluorescence (NIRF) imaging is a promising technique. Here we describe a novel, handheld, intraoperative NIRF camera system equipped with a 690 nm laser; we validated its utility in detecting and guiding resection of cancer tissues in two syngeneic rat models. The camera system was calibrated using an activated cathepsin-sensing probe (ProSense, VisEn Medical, Woburn, MA). Fluorescence intensity was strongly correlated with increased activated-probe concentration (R 2 = .997). During the intraoperative experiments, a camera exposure time of 10 ms was used, which provided the optimal tumor to background ratio. Primary mammary tumors (n = 20 tumors) were successfully resected under direct fluorescence guidance. The tumor to background ratio was 2.34 using ProSense680 at 10 ms camera exposure time. The background fluorescence of abdominal organs, in particular liver and kidney, was high, thereby limiting the ability to detect peritoneal metastases with cathepsin-sensing probes in these regions. In conclusion, we demonstrated the technical performance of this new camera system and its intraoperative utility in guiding resection of tumors.

Clinical prognostic value of combined analysis of Aldh1, Survivin, and EpCAM expression in colorectal cancer

Background:Tumour aggressiveness might be related to the degree of main cancer hallmark acquirement of tumour cells, reflected by expression levels of specific biomarkers. We investigated the expression of Aldh1, Survivin, and EpCAM, together reflecting main cancer hallmarks, in relation to clinical outcome of colorectal cancer (CRC) patients.Methods:Immunohistochemistry was performed using a tumour tissue microarray of TNM (Tumour, Node, Metastasis)-stage I–IV CRC tissues. Single-marker expression or their combination was assessed for associations with the clinical outcome of CRC patients (N=309).Results:Increased expression of Aldh1 or Survivin, or decreased expression of EpCAM was each associated with poor clinical outcome, and was therefore identified as clinically unfavourable expression. Analyses of the combination of all three markers showed worse clinical outcome, specifically in colon cancer patients, with an increasing number of markers showing unfavourable expression. Hazard ratios ranged up to 8.3 for overall survival (P<0.001), 36.6 for disease-specific survival (P<0.001), and 27.1 for distant recurrence-free survival (P<0.001).Conclusions:Our data identified combined expression levels of Aldh1, Survivin, and EpCAM as strong independent prognostic factors, with high hazard ratios, for survival and tumour recurrence in colon cancer patients, and therefore reflect tumour aggressiveness.

A prolactin-dependent, metastasising rat mammary carcinoma as a model for endocrine-related tumour dormancy

In order to study the growth kinetics of breast tumours during long-term hormonal withdrawal, we developed a transplantable, invasive mammary carcinoma EMR-86 that originated in a female WAG/Olac rat bearing a subcutaneously implanted oestrogen pellet (EP). Outgrowth of transplanted tumours occurs only in the presence of an EP, and metastases are formed in lungs and regional lymph nodes. Subsequent EP removal induces rapid regression. However, tumours do not disappear completely, as small nodules persist. These dormant tumour remnants can be restimulated even after long periods. Because EP-stimulated tumours regressed after treatment with bromocriptine and dormant tumours in non-oestrogenized rats grew out after treatment with perphenazine, prolactin is the major growth-stimulating hormone in this model. Cell kinetics in the growing, regressing and dormant phase were studied by immunocytochemical detection of DNA-incorporated bromodeoxyuridine (BrdUrd) in tissue sections. BrdUrd labelling indices decreased from 21.6 +/- 3.0% to less than 1% within 7 days after EP removal. After prolonged hormonal withdrawal (up to 90 days) BrdUrd-labelled tumour cells could always be demonstrated (range 0.4-0.8%), without a concomitant increase in tumour volume. Additional treatment either with bromocriptine or with ovariectomy could not significantly reduce this residual proliferative activity, as demonstrated by continuous BrdUrd labelling experiments. The results indicate that in vivo dormancy may represent a steady state of cell division and cell loss, rather than an accumulation of cells in a non-cycling G0 state.

Surgically induced cytokinetic responses in experimental rat mammary tumor models.

The effect of surgical removal of “primary” tumors on the cytokinetics of local tumor remnants, secondary implants, and metastases was investigated in three different rat tumor models in the Wag/Rij rat: a slow‐growing (MCR83) and a fast‐growing (EMR86) hormone‐dependent mammary tumor and a rapidly, but autonomously growing carcinoma (MCR86). The latter two tumors had metastatic potential. Cell kinetic studies were done using in vivo labeling with 5′ ‐ bromodeoxyuridine (BrdUrd). Thirty‐three hours after removal of a subcutaneous MCR83 flank tumor, secondary implants showed a significant (P < 0.05) but transient increase in the BrdUrd labeling index (LI). A more rapid and prolonged increase, lasting for at least 7 days, was observed in EMR86 lymph node and lung metastases. In both models, no effect was observed after sham surgery (consisting of opening and closing of the skin under anesthesia). Removal of MCR86 tumors (growing in the hind leg muscle) also resulted in a rapid, transient LI increase in metastases. Continuous BrdUrd labeling experiments in this tumor model did not favor the hypothesis that the LI increase predominantly resulted from an increase in the growth fraction. Moreover, in this model, the effect was related to operation trauma. A similar increase in LI, although smaller than after tumor removal, was seen after major surgical trauma in MCR83 flank tumors. These results indicate that in the rat, tumor removal and/or major surgical trauma may modulate the cytokinetics of distant metastases significantly. A study of the systemic, possibly endocrine, factors involved in the growth‐stimulating effect of surgical trauma in these rat tumor models may help to assess the clinical relevance of these findings for patients with breast cancer.

Effect of hormone depletion on cell survival in the EMR-86 rat mammary carcinoma.

Growth of the transplantable EMR-86 rat mammary carcinoma depends on elevated prolactin levels which are induced by oestrogenic stimulation of the pituitary. We investigated histological and cell kinetic changes during tumour regression after removal of implanted oestrogen pellets (EP), and we especially focused on the role of apoptosis. After EP removal, serum prolactin decreased to basal levels in 5 days, reaching its largest depletion during the first day. Similarly, S-phase cell fractions, assessed by bromodeoxyuridine (BrdUrd) incorporation, decreased to half the initial value during the first day and developed into a gradual decrease to basal levels thereafter. Within 10 days, tumour volumes were reduced to 20% without striking changes in tissue architecture. To quantify apoptosis, we applied a method that stains DNA breaks in tissue sections and subsequently measured the stained area by automated image cytometry. This procedure was necessary, as the subtle changes could not be detected by histological examination alone. One day after the rapid decline of the S-phase fraction, a 3-fold increase in apoptotic area was observed that remained for about 3 days and then gradually decreased. This correlated with the histologically observed reduction of tumour cells. In spite of the major cell loss, regression came to a halt after about 10 days. The surviving cell fraction is discussed within the context of a stem cell hypothesis, in which tumour cells with stem cell characteristics are less susceptible to hormone-induced apoptosis than their (non-stem) daughter cells. This notion has implications for the eradication of residual tumour cells, because a diminished susceptibility might also apply to apoptosis induced by radio- or chemotherapy.

Intra-Operative Imaging of the Invasive Tumor Border by a Cathepsin-Sensing Near-Infrared Fluorescence Probe in a Syngeneic Breast Cancer Rat Model.

Background: An incomplete tumor resection occurs in 5-45% of the patients undergoing breast-conserving surgery. As the surgeon can only rely on palpation and visual inspection, real-time visualization of cancer cells at the time of surgery is needed to increase the number of complete tumor resections. Near-infrared fluorescence (NIRF) imaging is a promising technique to assess the extent of disease during surgery. This study aimed to validate NIRF imaging in a breast cancer rat model.Methods: The hormone-dependent syngeneic breast cancer rat model EMR86 and its derived cell line MCR86 were used. Tumor cells were detected with the cathepsin-activatable (mainly cathepsin-B) NIRF probe ProSense® (VisEn Medical, USA). Fluorescence imaging of the animals, organs and tissue sections was performed using IVIS Spectrum (Caliper, USA), Odyssey (LI-COR, USA) and SP5 microscope (Leica, Germany). Intra-operative fluorescence imaging was performed using the Fluobeam700 system equipped with a 690 nm class 3B laser and a CCD camera (Fluoptics, France).Results: ProSense was activated by cleavage in MCR86 cells. The signal intensity was linearly correlated with the number of cells, the concentration of ProSense and the incubation time (all R 2 >0.93, p 3 ). In contrast to the in vitro data, no difference in fluorescence intensity was found between 24hr and 48hr after injection of ProSense (paired t=-0.27, p=0.80). Therefore, subsequent experiments were conducted 24hr after injection. Calibration of the intra-operative camera system demonstrated a strong correlation of fluorescence intensity and activated ProSense concentration (R 2 =0.9903). Using the intra-operative NIRF camera, all 64 mammary tumors (0.01-1.8 cm 3 ) were successfully detected. Histological assessment of residual fluorescent hotspots confirmed the presence of breast cancer cells indicating incomplete resections of the primary tumor (margin positivity). The signal intensity of the tumor was 60-fold higher than the surrounding tissue (p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5007.