Calreticulin (CRT) is a 46 kDa endoplasmic reticulum (ER)–resident chaperone protein that plays a critical role in calcium homeostasis and glycoprotein folding (Michalak et al., 1999). In addition to these canonical ER functions, CRT can become detectable at the plasma membrane under conditions of cellular stress, where it functions as a damage-associated molecular pattern (DAMP) involved in immune recognition and clearance of stressed or damaged cells (Gardai et al., 2005; Obeid et al., 2007). Surface-exposed CRT has been described in several malignancies, including bladder, lung, breast, and ovarian cancers, where it has been associated with altered cellular signaling and immune engagement (Kageyama et al., 2004; Liu et al., 2012; Song et al., 2012). In prostate cancer, however, reports of CRT expression and localization have been inconsistent, with both increased and decreased expression described depending on disease context (Alur et al., 2009). These discrepancies highlight the need for reproducible and quantitative approaches to evaluate CRT localization in prostate cancer models.

CRT contains three structural domains (N, P, and C), including a C-terminal Lys-Asp-Glu-Leu (KDEL) sequence that mediates ER retention through receptor-dependent retrieval pathways (Cabrera et al., 2003; Michalak et al., 1999). Despite this retention mechanism, CRT has been observed at the cell surface under conditions of cellular stress, suggesting that redistribution from the ER can occur. The processes governing CRT redistribution remain incompletely defined and may involve multiple cellular pathways, including ER stress responses and post-translational modifications. Proteomic studies have demonstrated that ER-resident proteins can undergo lysine acetylation (Pehar et al., 2012), and altered acetylation profiles have been observed in prostate cancer cells (Pathak et al., 2015). These findings provide biological context for evaluating CRT localization under pharmacologic conditions that influence cellular stress and acetylation state, although the present study does not directly assess these mechanisms.

Quantification of surface-exposed CRT (ectoCRT) in prostate cancer models presents several technical challenges. LNCaP cells exhibit relatively low baseline surface CRT expression, making detection sensitive to staining conditions and imaging parameters. Variability in fluorescence intensity measurements can arise from differences in antibody accessibility, imaging exposure, and segmentation approaches. Furthermore, many studies of protein trafficking rely on live-cell imaging or time-lapse microscopy, which may not be accessible in all research environments. These limitations underscore the need for a standardized, reproducible fixed-cell imaging workflow that enables reliable distinction between intracellular and surface-accessible CRT pools.

In this study, we optimized a non-permeabilized immunofluorescence protocol to detect surface CRT in LNCaP cells using total anti-CRT antibody staining, with complementary experiments performed in cells transfected with wild-type CRT from the pMSCV-IRES-GFP/CALR wt plasmid. Surface staining was performed by incubating cells with primary antibody prior to fixation, restricting antibody access to extracellular epitopes. Intracellular CRT detection required permeabilization using 1% Triton X-100 in phosphate-buffered saline (PBS). Membrane integrity under non-permeabilized conditions was validated using ERp57 as an intracellular control protein, which showed no detectable signal in the absence of permeabilization, confirming that intracellular epitopes remained inaccessible during surface staining.

Cells were treated with thapsigargin (TG), a well-established ER stress inducer known to influence CRT cell surface translocation (Jeffery et al., 2011), as a positive control condition. Comparative treatment with trichostatin A (TSA), a potent histone deacetylase (HDAC) inhibitor (Frønsdal et al., 2004), was used to evaluate increased acetylation conditions on CRT trafficking. Given that altered acetylation profiles are observed in prostate cancer (Watson, et al., 2010), we utilized TSA to test whether increased global acetylation influences CRT surface mobilization, serving as a secondary stress-response model alongside TG. 0.1% DMSO was included as a vehicle control. TG and TSA concentrations were determined by preliminary Methyl Thiazolyl Tetrazolium (MTT) optimization assays and supporting literature on ER stress induction and HDAC inhibition in prostate cancer models. Conditions were chosen that produced detectable CRT redistribution while not compromising cell integrity. Under TG treatment conditions, a visible redistribution of CRT signal toward the cell periphery was observed relative to vehicle-treated controls. While intracellular CRT fluorescence intensity decreased and surface-associated signal increased, direct comparison of these measures revealed weak correlation across treatment conditions. These findings indicate that absolute fluorescence intensity measurements alone may not fully capture the extent of CRT redistribution.

To address this limitation, we implemented a redistribution factor (RF) defined as ectoCRT/(ectoCRT + endoCRT), which quantifies the relative partitioning of CRT between intracellular and surface compartments within individual cells. By normalizing surface-associated signal to total detectable CRT, RF provides a metric that is less influenced by global changes in protein expression or imaging variability. This approach is particularly useful in systems with low baseline surface expression, where small changes in fluorescence intensity may be difficult to interpret using absolute measurements alone. Across independent experiments and standardized imaging conditions, RF values demonstrated reproducibility and sensitivity to treatment-dependent changes in CRT localization.

Importantly, the present study does not directly evaluate the molecular mechanisms underlying CRT redistribution but instead establishes a reproducible imaging and quantification framework for assessing CRT partitioning in prostate cancer cells. This fixed-cell approach eliminates the requirement for live-cell imaging systems and enables broader application in laboratories equipped with standard fluorescence microscopy platforms.

In summary, we describe a standardized non-permeabilized immunofluorescence workflow and a redistribution-based quantification strategy for evaluating ER-to-surface CRT mobilization in LNCaP prostate cancer cells. This method provides a practical and reproducible approach for measuring CRT redistribution and may facilitate future studies investigating protein trafficking, cellular stress responses, and DAMP-associated signaling in cancer models.

Cell Culture and Transfection

LNCaP human prostate adenocarcinoma cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin under standard culture conditions (37°C, 5% CO₂). For transfection studies, cells were transiently transfected with GFP-tagged calreticulin (pMSCV-IRES-GFP/CALR wt plasmid - a gift from Norio Komatsu) and incubated for 24 hours prior to treatment. GFP fluorescence was used as the primary readout for total CRT expression across all experimental conditions.

Drug Treatments

Following drug administration, cells were incubated for 4 hours for endogenous CRT experiments or 24 hours for transfected (GFP-CRT) experiments with:

These conditions were selected to evaluate CRT redistribution under pharmacologic perturbation.

Immunofluorescence Staining

Surface CRT (ectoCRT) Staining:

Surface-accessible CRT was detected under non-permeabilized conditions. Cells were incubated with either anti-CRT (for endogenous CRT experiments) or anti-GFP (for transfection experiments) primary antibody prior to fixation to restrict antibody access to extracellular epitopes. Following primary antibody incubation, cells were fixed with 2% paraformaldehyde (PFA) and subsequently incubated with fluorophore-conjugated secondary antibodies for 1 hour at room temperature. Nuclei were stained with DAPI.

Membrane integrity was validated using ERp57 as an intracellular control protein, which showed no detectable signal under non-permeabilized conditions, confirming that intracellular epitopes remained inaccessible.

Intracellular CRT Staining:

For intracellular CRT detection, cells were fixed with 4% paraformaldehyde (PFA), washed with 0.01 M phosphate-buffered saline (PBS), and permeabilized using 1% Triton X-100 in PBS. Cells were then blocked in normal donkey serum and incubated with either anti-CRT or anti-GFP primary antibody overnight at 4°C. Following washing, cells were incubated with fluorophore-conjugated secondary antibodies and counterstained with DAPI.

Image Acquisition

Fluorescence images were acquired using identical imaging settings across all treatment conditions to ensure comparability. Exposure time was fixed at 1 ms for all channels and samples to minimize variability in fluorescence intensity measurements.

Image Analysis and Quantification

Image analysis was performed using CellProfiler on 16 bit images with custom pipelines designed for single-cell segmentation and fluorescence quantification.

Background fluorescence was quantified from cell-free regions outside segmented cell regions of interest (ROIs) within each image. The average background fluorescence intensity was subtracted from the mean fluorescence intensity of each ROI to generate corrected CRT intensity measurements. Area-based and intensity-based filtering were then applied to exclude debris and incorrectly segmented objects before calculation of fluorescence intensity and redistribution factor (RF) values.

For endogenous experiments, approximately 30 cells were analyzed for surface CRT and 50 cells for intracellular CRT per condition. For GFP-transfected experiments, approximately 23 cells were analyzed for surface CRT and 99 cells for intracellular CRT per condition. All analyses were performed across three independent experiments.

Experimental conditions were validated in parallel using both endogenous staining (anti-CRT) and transfected reporters (anti-GFP) to confirm consistency.

Use of a single GFP-CRT reporter for transfection experiments across all conditions minimized inter-channel variability and enabled direct comparison of CRT redistribution between treatments.

Redistribution Factor Calculation

Background-subtracted mean fluorescence intensity values were used to calculate a redistribution factor (RF) for each cell, defined as:

RF= ectoCRT /ectoCRT + endoCRT

ectoCRT = surface CRT and endoCRT = intracellular CRT

This metric represents the relative partitioning of CRT between surface-accessible and intracellular compartments.

Statistical Analysis

Fluorescence intensity measurements were obtained at the single-cell level for both intracellular and surface conditions. Statistical comparisons between treatment groups were performed using unpaired two-tailed Student’s t-tests.

Comparisons were conducted between vehicle (0.1% DMSO) and treatment conditions (thapsigargin [TG] or trichostatin A [TSA]) for:

Sample sizes varied by experiment: for endogenous experiments, approximately 30 cells were analyzed for surface CRT and 50 cells for intracellular CRT per condition. For GFP-transfected experiments, approximately 23 cells were analyzed for surface CRT and 99 cells for intracellular CRT per condition. All analyses were conducted across three independent experiments.

Data are presented as mean ± standard deviation. Statistical significance values (p) and effect sizes (Cohen’s d) are reported in the figure.