pH-Responsive Polymers for Improving the Signal-to-Noise Ratio of Hypoxia PET Imaging with [18 F]Fluoromisonidazole.

To improve the signal-to-noise ratio of hypoxia positron emission tomography (PET) imaging, stimuli-responsive polymers are designed for the delivery of the hypoxia PET tracer fluorine-18 labeled fluoromisonidazole ([18 F]FMISO). Linear poly(N-(2-(hydroxypropyl)methacrylamide)) polymers are functionalized with hydrazide linkers that form pH-sensitive acyl hydrazone bonds after their conjugation to an [18 F]FMISO ketone analogue. The release of the [18 F]FMISO ketone analogue from the polymers is considerably faster at a lower pH and its uptake is significantly higher in cancer cells growing under acidic conditions. Additionally, the retention of the PET tracer is significantly higher in hypoxic cells compared to normoxic cells. The delivery of a PET tracer using stimuli-responsive polymers may be an attractive strategy to improve signal-to-noise ratios in PET imaging.

Loss of KCNQ1 expression in stage II and stage III colon cancer is a strong prognostic factor for disease recurrence.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer worldwide. Accurately identifying stage II CRC patients at risk for recurrence is an unmet clinical need. KCNQ1 was previously identified as a tumour suppressor gene and loss of expression was associated with poor survival in patients with CRC liver metastases. In this study the prognostic value of KCNQ1 in stage II and stage III colon cancer patients was examined. METHODS: KCNQ1 mRNA expression was assessed in 90 stage II colon cancer patients (AMC-AJCCII-90) using microarray gene expression data. Subsequently, KCNQ1 protein expression was evaluated in an independent cohort of 386 stage II and stage III colon cancer patients by immunohistochemistry of tissue microarrays. RESULTS: Low KCNQ1 mRNA expression in stage II microsatellite stable (MSS) colon cancers was associated with poor disease-free survival (DFS) (P=0.025). Loss of KCNQ1 protein expression from epithelial cells was strongly associated with poor DFS in stage II MSS (P<0.0001), stage III MSS (P=0.0001) and stage III microsatellite instable colon cancers (P=0.041). KCNQ1 seemed an independent prognostic value in addition to other high-risk parameters like angio-invasion, nodal stage and microsatellite instability-status. CONCLUSIONS: We conclude that KCNQ1 is a promising biomarker for prediction of disease recurrence and may aid stratification of patients with stage II MSS colon cancer for adjuvant chemotherapy.

Glucose Transporter 1 (SLC2A1) and Vascular Endothelial Growth Factor A (VEGFA) Predict Survival After Resection of Colorectal Cancer Liver Metastasis.

OBJECTIVE: To investigate the individual and combined prognostic value of HIF1α, SLC2A1, and vascular endothelial growth factor A (VEGFA) in a multi-institutional cohort of patients with resected colorectal cancer liver metastasis (CRCLM). BACKGROUND: In the majority of patients with CRCLM, resection seems not to be curative, despite its curative intent. Overexpression of hypoxia-inducible factor 1α (HIF1α), glucose transporter 1 (SLC2A1; also known as GLUT1), and VEGFA has been associated with tumor progression and poor prognosis of patients with colorectal cancer (CRC). METHODS: Tissue microarrays were generated using CRCLM and patient-matched primary CRC from patients who underwent CRCLM resection between 1990 and 2010. Prognostic value of HIF1α, SLC2A1, and VEGFA was determined by immunohistochemistry. A 500-fold cross-validated hazard rate ratio (HRRav) for overall survival was calculated. RESULTS: HIF1α, SLC2A1, and VEGFA expression could be evaluated in 328, 350, and 335 patients, respectively. High SLC2A1 expression was associated with good prognosis (HRRav, 0.67; P (HRR >1)  < 0.01) and high VEGFA expression to poor prognosis (HRRav, 1.84; P (HRR < 1)  = 0.02), also after multivariate analysis including established clinicopathological prognostic variables (HRRav, 0.67; P (HRR > 1)  < 0.01 and HRRav, 1.50; P (HRR < 1)  = 0.02, respectively). SLC2A1 showed prognostic value particularly in patients treated with systemic therapy (P < 0.01), whereas the prognostic value of VEGFA expression was mainly observed in patients not treated with systemic therapy (P < 0.01). Prognosis was especially poor in patients with both low SLC2A1 and high VEGFA expression (P < 0.01). HIF1α expression was not associated with survival. CONCLUSIONS: SLC2A1 and VEGFA expression are prognostic molecular biomarkers for patients with CRCLM with added value to established clinicopathological variables.

Combination of episulfide ring-opening polymerization with ATRP for the preparation of amphiphilic block copolymers.

We report for the first time the combination of ATRP and ring-opening episulfide polymerization as a means to synthesize polysulfide-based low-dispersity amphiphilic block copolymers. The most significant finding is the possibility to perform ATRP under mild conditions using poly(propylene sulfide) macroinitiators, apparently without any significant copper sequestration by the polysulfides. Using glycerol monomethacrylate (GMMA) as a hydrophilic monomer, the polymers self-assembled in colloidal structures with a morphology depending on the PS/GMMA ratio, but also probably on GMMA degree of polymerization. We here also present a new AFM-based method to calculate the average number of amphiphilic macromolecules per micelle.

Comparative in vivo biodistribution of cells labelled with [89Zr]Zr-(oxinate)4 or [89Zr]Zr-DFO-NCS using PET.

BACKGROUND: In vivo monitoring of cell biodistribution using positron emission tomography (PET) provides a quantitative non-invasive method to further optimize cell therapies and related new developments in the field. Our group has earlier optimized and evaluated the in vitro properties of two radiotracers,[89Zr]Zr-(oxinate)4 and [89Zr]Zr-DFO-NCS, for the radiolabelling of different cell types. Here, we performed a microPET study to assess the in vivo biodistribution of cells in rats using these two radiotracers. Human decidual stromal cells (hDSC) and rat macrophages (rMac) were radiolabelled with [89Zr]Zr-(oxinate)4 or [89Zr]Zr-DFO-NCS. Rats were intravenously injected with radiolabelled cells, and the in vivo biodistribution was monitored with microPET/CT imaging for up to day 7. Organ uptake was evaluated and presented as a percentage of injected activity per gram tissue (%IA/g) and total absorbed organ doses (mSv/MBq). RESULTS: The biodistribution in vivo showed an immediate uptake in the lungs. Thereafter, [89Zr]Zr-(oxinate)4 labelled cells migrated to the liver, while the signal from [89Zr]Zr-DFO-NCS labelled cells lingered in the lungs. The differences in the in vivo behaviour for the same cell type appeared related to the radiotracer labelling. After 24 h, [89Zr]Zr-(oxinate)4 labelled cells had over 70% higher liver uptake for both hDSC and rMac compared to [89Zr]Zr-DFO-NCS labelled cells, whereas [89Zr]Zr-DFO-NCS labelled cells showed over 60% higher uptake in the lungs compared to [89Zr]Zr-(oxinate)4 labelled cells. This difference in both lung and liver uptake continued until day 7. Dosimetry calculations showed a higher effective dose (mSv/MBq) for [89Zr]Zr-DFO-NCS compared to [89Zr]Zr-(oxinate)4, for both cell types. Although the bone uptake was higher for [89Zr]Zr-(oxinate)4 labelled cells, the prolonged uptake in the lungs contributed to a significant crossfire to bone marrow resulting in a higher bone dose. CONCLUSION: The [89Zr]Zr-DFO-NCS labelled cells suggest a prolonged accumulation in the lungs, while [89Zr]Zr-(oxinate)4 suggests quicker clearance of the lungs followed by accumulation in the liver. Accumulation of radiolabelled cells in the liver corresponds to other cell-tracking methods. Further studies are required to determine the actual location of the [89Zr]Zr-DFO-NCS labelled cell.

Optimisation of the Synthesis and Cell Labelling Conditions for [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS: a Direct In Vitro Comparison in Cell Types with Distinct Therapeutic Applications.

BACKGROUND: There is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells in vivo is often impossible due to unreliable methods. Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [89Zr]Zr-oxine (8-hydroxyquinoline) and [89Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers. PROCEDURES: Several parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [89Zr]Zr-oxine or [89Zr]Zr-DFO-NCS. The cellular retention of 89Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining. RESULTS: The optimised synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. 89Zr labelling neither significantly affected the cell viability (cell viability loss was in the range of 1-8 % compared to its corresponding non-labelled cells, P value > 0.05) nor the cells' proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling. CONCLUSIONS: Our study demonstrates that [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [89Zr]Zr-oxine was superior to [89Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency.

Expression of the immune modulator secretory leukocyte protease inhibitor (SLPI) in colorectal cancer liver metastases and matched primary tumors is associated with a poorer prognosis.

Secretory leukocyte protease inhibitor (SLPI), a pleiotropic protein expressed by healthy intestinal epithelial cells, functions as an inhibitor of NF-κB and neutrophil proteases and exerts antimicrobial activity. We previously showed SLPI suppresses intestinal epithelial chemokine production in response to microbial contact. Increased SLPI expression was recently detected in various types of carcinoma. In addition, accumulating evidence indicates SLPI expression is favorable for tumor cells. In view of these findings and the abundance of SLPI in the colonic epithelium, we hypothesized SLPI promotes colorectal cancer (CRC) growth and metastasis. Here, we aimed to establish whether SLPI expression in CRC is related to clinical outcome. Using a cohort of 507 patients with CRC who underwent resection of liver metastases, we show that high SLPI protein expression in both liver metastases and primary CRC is associated with significantly shorter overall survival after resection of liver metastases. The prognostic value of SLPI in CRC patients with liver metastases implies a role for SLPI in the formation of metastasis of human CRC. Based on the immune regulatory functions of SLPI, we anticipate that expression of SLPI provides tumors with a mechanism to evade infiltration by immune cells.

Design and preclinical evaluation of nanostars for the passive pretargeting of tumor tissue.

INTRODUCTION: Pretargeting strategies that do not rely on the expression of molecular targets have expanded imaging and therapy options for cancer patients. Nanostars with designed multivalency and which highly accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect may therefore be the ideal vectors for the development of a passive pretargeting approach. METHODS: Nanostars were synthesized, consisting of 7-8 center-cross-linked arms that were modified with trans-cyclooctene (TCO) using poly(ethylene glycol) (PEG) linkers of 12 or 106 monomer units or without linker. The bioorthogonal click reaction with radiofluorinated 2,2'-(7-(2-(tetrazine-poly(ethyleneglycol)11-amino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ([18F]F-Tz-PEG11-NODA) or 2,2'-(7-(2-(tetrazine-amino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ([18F]F-Tz-NODA) was measured by ex vivo biodistribution studies and positron emission tomography (PET) in mice bearing tumors with high EPR characteristics. Bioorthogonal masking was performed using a tetrazine-functionalized dextran polymer (Tz-DP). RESULTS: Highest tumor accumulation of [18F]F-Tz-PEG11-NODA was observed for nanostars functionalized with TCO without linker, with a tumor uptake of 3.2 ± 0.4%ID/g and a tumor-to-muscle ratio of 12.8 ± 4.2, tumor-to-large intestine ratio of 0.5 ± 0.3 and tumor-to-kidney ratio of 2.0 ± 0.3, being significantly higher than for nanostars functionalized with TCO-PEG12 (P < 0.05) or TCO-PEG106 (P < 0.05). Tumor uptake and tumor-to-tissue ratios did not improve upon bioorthogonal masking with Tz-DP or when using a smaller, more lipophilic tetrazine([18F]F-Tz-NODA). CONCLUSIONS: A pretargeting strategy was developed based on the passive delivery of TCO-functionalized nanostars. Such a strategy would allow for the imaging and treatment of tumors with apparent EPR characteristics, with high radioactive tumor doses and minimal doses to off-target tissues.

Delivery of polymeric nanostars for molecular imaging and endoradiotherapy through the enhanced permeability and retention (EPR) effect.

Expression levels of biomarkers are generally unknown at initial diagnosis. The development of theranostic probes that do not rely on biomarker availability would expand therapy options for cancer patients, improve patient selection for nanomedicine and facilitate treatment of inoperable patients or patients with acquired therapy resistance. Herein, we report the development of star polymers, also known as nanostars, that allow for molecular imaging and/or endoradiotherapy based on passive targeting via the enhanced permeability and retention (EPR) effect. Methods: We synthesised a star copolymer, consisting of 7-8 centre-cross-linked arms that were modified with Gd3+ for magnetic resonance imaging (MRI), and functionalised either with 89Zr for in vivo quantification and positron emission tomography (PET) imaging, or with 177Lu for endoradiotherapy. 1H longitudinal relaxivities were determined over a continuum of magnetic field strengths ranging from 0.24 mT - 0.94 T at 37 °C (nuclear magnetic relaxation dispersion (NMRD) profile) and T1-weighted MRI contrast enhancement was visualized at 3 T and 7 T. PET imaging and ex vivo biodistribution studies were performed in mice bearing tumours with high EPR (CT26) or low EPR (BxPC3) characteristics. Therapy studies were performed in mice with high EPR tumours and mean absorbed organ doses were estimated for a standard human model. Results: The star copolymer with Gd3+ displayed a significantly superior contrast enhancement ability (T1 = 0.60 s) compared to the standard clinical contrast agent Gadovist (T1 = 1.0 s). Quantification of tumour accumulation using the radiolabelled nanostars in tumour-bearing mice demonstrated an exceptionally high uptake in tumours with high EPR characteristics (14.8 - 21.7 %ID/g). Uptake of the star polymers in tumours with low EPR characteristics was significantly lower (P<0.001), suggesting passive tumour accumulation of the nanostars via the EPR effect. Survival of mice treated with high dose 177Lu-labelled star polymers was significantly higher than survival of mice treated with lower therapy doses or control mice (P=0.001), demonstrating the utility of the 177Lu-labelled star polymers as platforms for endoradiotherapy. Conclusion: Our work highlights the potential of star polymers as probes for the molecular imaging of cancer tissue or for the passive delivery of radionuclides for endoradiotherapy. Their high functionalisability and high tumour accumulation emphasises their versatility as powerful tools for nanomedicine.

Temporal Modulation of HER2 Membrane Availability Increases Pertuzumab Uptake and Pretargeted Molecular Imaging of Gastric Tumors.

Human epidermal growth factor receptor 2 (HER2) is used as a tumor biomarker and therapeutic target. Pertuzumab is an anti-HER2 antibody, and its binding to tumor cells requires HER2 to be present at the cell membrane. However, the cellular distribution of HER2 protein in gastric tumors is dynamic, and HER2 internalization decreases antibody binding to tumor cells. These features preclude the use of pretargeted strategies for molecular imaging and therapy. We explored the pharmacological modulation of HER2 endocytosis as a strategy to improve pertuzumab uptake in HER2-positive gastric tumors and allow the use of a pretargeted imaging approach. Methods: We conducted in vitro and in vivo studies with NCI-N87 gastric cancer cells to determine how HER2 endocytosis affects pertuzumab binding to tumor cells. Lovastatin, a clinically approved cholesterol-lowering drug, was used to modulate caveolae-mediated HER2 endocytosis. Results: Administration of lovastatin to NCI-N87 cancer cells resulted in significant accumulation of non-activated HER2 dimers at the cell surface. Pretreatment of NCI-N87 cells with lovastatin increased in vitro specific accumulation of membrane-bound 89Zr-labeled pertuzumab. Lovastatin-enhanced pertuzumab tumor uptake was also observed in NCI-N87 gastric cancer xenografts, allowing tumor detection as early as 4 h and high-contrast images at 48 h after tracer administration via PET. Temporal enhancement of HER2 membrane availability by lovastatin allowed imaging of cell surface HER2 with transcyclooctene-conjugated antibodies and 18F-labeled tetrazine. Conclusion: Temporal pharmacological modulation of membrane HER2 may be clinically relevant and exploitable for pretargeted molecular imaging and therapy in gastric tumors.

Biologically Targeted Magnetic Hyperthermia: Potential and Limitations.

Hyperthermia, the mild elevation of temperature to 40-43°C, can induce cancer cell death and enhance the effects of radiotherapy and chemotherapy. However, achievement of its full potential as a clinically relevant treatment modality has been restricted by its inability to effectively and preferentially heat malignant cells. The limited spatial resolution may be circumvented by the intravenous administration of cancer-targeting magnetic nanoparticles that accumulate in the tumor, followed by the application of an alternating magnetic field to raise the temperature of the nanoparticles located in the tumor tissue. This targeted approach enables preferential heating of malignant cancer cells whilst sparing the surrounding normal tissue, potentially improving the effectiveness and safety of hyperthermia. Despite promising results in preclinical studies, there are numerous challenges that must be addressed before this technique can progress to the clinic. This review discusses these challenges and highlights the current understanding of targeted magnetic hyperthermia.

Molecular imaging of aurora kinase A (AURKA) expression: Synthesis and preclinical evaluation of radiolabeled alisertib (MLN8237).

INTRODUCTION: Survival of patients after resection of colorectal cancer liver metastasis (CRCLM) is 36%-58%. Positron emission tomography (PET) tracers, imaging the expression of prognostic biomarkers, may contribute to assign appropriate management to individual patients. Aurora kinase A (AURKA) expression is associated with survival of patients after CRCLM resection. METHODS: We synthesized [(3)H]alisertib and [(11)C]alisertib, starting from [(3)H]methyl nosylate and [(11)C]methyl iodide, respectively. We measured in vitro uptake of [(3)H]alisertib in cancer cells with high (Caco2), moderate (A431, HCT116, SW480) and low (MKN45) AURKA expression, before and after siRNA-mediated AURKA downmodulation, as well as after inhibition of P-glycoprotein (P-gp) activity. We measured in vivo uptake and biodistribution of [(11)C]alisertib in nude mice, xenografted with A431, HCT116 or MKN45 cells, or P-gp knockout mice. RESULTS: [(3)H]Alisertib was synthesized with an overall yield of 42% and [(11)C]alisertib with an overall yield of 23%±9% (radiochemical purity ≥99%). Uptake of [(3)H]alisertib in Caco2 cells was higher than in A431 cells (P=.02) and higher than in SW480, HCT116 and MKN45 cells (P<.01). Uptake in A431 cells was higher than in SW480, HCT116 and MKN45 cells (P<.01). Downmodulation of AURKA expression reduced [(3)H]alisertib uptake in Caco2 cells (P<.01). P-gp inhibition increased [(3)H]alisertib uptake in Caco2 (P<.01) and MKN45 (P<.01) cells. In vivo stability of [(11)C]alisertib 90min post-injection was 94.7%±1.3% and tumor-to-background ratios were 2.3±0.8 (A431), 1.6±0.5 (HCT116) and 1.9±0.5 (MKN45). In brains of P-gp knockout mice [(11)C]alisertib uptake was increased compared to uptake in wild-type mice (P<.01) CONCLUSIONS: Radiolabeled alisertib can be synthesized and may have potential for the imaging of AURKA, particularly when AURKA expression is high. However, the exact mechanisms underlying alisertib accumulation need further investigation. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Radiolabeled alisertib may be used for non-invasively measuring AURKA protein expression and to stratify patients for treatment accordingly.

A prognostic classifier for patients with colorectal cancer liver metastasis, based on AURKA, PTGS2 and MMP9.

BACKGROUND: Prognosis of patients with colorectal cancer liver metastasis (CRCLM) is estimated based on clinicopathological models. Stratifying patients based on tumor biology may have additional value. METHODS: Tissue micro-arrays (TMAs), containing resected CRCLM and corresponding primary tumors from a multi-institutional cohort of 507 patients, were immunohistochemically stained for 18 candidate biomarkers. Cross-validated hazard rate ratios (HRRs) for overall survival (OS) and the proportion of HRRs with opposite effect (P(HRR < 1) or P(HRR > 1)) were calculated. A classifier was constructed by classification and regression tree (CART) analysis and its prognostic value determined by permutation analysis. Correlations between protein expression in primary tumor-CRCLM pairs were calculated. RESULTS: Based on their putative prognostic value, EGFR (P(HRR < 1) = .02), AURKA (P(HRR < 1) = .02), VEGFA (P(HRR < 1) = .02), PTGS2 (P(HRR < 1) = .01), SLC2A1 (P(HRR > 1) < 01), HIF1α (P(HRR > 1) = .06), KCNQ1 (P(HRR > 1) = .09), CEA (P (HRR > 1) = .05) and MMP9 (P(HRR < 1) = .07) were included in the CART analysis (n = 201). The resulting classifier was based on AURKA, PTGS2 and MMP9 expression and was associated with OS (HRR 2.79, p < .001), also after multivariate analysis (HRR 3.57, p < .001). The prognostic value of the biomarker-based classifier was superior to the clinicopathological model (p = .001). Prognostic value was highest for colon cancer patients (HRR 5.71, p < .001) and patients not treated with systemic therapy (HRR 3.48, p < .01). Classification based on protein expression in primary tumors could be based on AURKA expression only (HRR 2.59, p = .04). CONCLUSION: A classifier was generated for patients with CRCLM with improved prognostic value compared to the standard clinicopathological prognostic parameters, which may aid selection of patients who may benefit from adjuvant systemic therapy.

MGL ligand expression is correlated to BRAF mutation and associated with poor survival of stage III colon cancer patients.

Colorectal cancer (CRC) is the third most prevalent cancer type worldwide with a mortality rate of approximately 50%. Elevated cell-surface expression of truncated carbohydrate structures such as Tn antigen (GalNAcα-Ser/Thr) is frequently observed during tumor progression. We have previously demonstrated that the C-type lectin macrophage galactose-type lectin (MGL), expressed by human antigen presenting cells, can distinguish healthy tissue from CRC through its specific recognition of Tn antigen. Both MGL binding and oncogenic BRAF mutations have been implicated in establishing an immunosuppressive microenvironment. Here we aimed to evaluate whether MGL ligand expression has prognostic value and whether this was correlated to BRAF(V600E) mutation status. Using a cohort of 386 colon cancer patients we demonstrate that high MGL binding to stage III tumors is associated with poor disease-free survival, independent of microsatellite instability or adjuvant chemotherapy. In vitro studies using CRC cell lines showed an association between MGL ligand expression and the presence of BRAF(V600E). Administration of specific BRAF(V600E) inhibitors resulted in decreased expression of MGL-binding glycans. Moreover, a positive correlation between induction of BRAF(V600E) and MGL binding to epithelial cells of the gastrointestinal tract was found in vivo using an inducible BRAF(V600E) mouse model. We conclude that the BRAF(V600E) mutation induces MGL ligand expression, thereby providing a direct link between oncogenic transformation and aberrant expression of immunosuppressive glycans. The strong prognostic value of MGL ligands in stage III colon cancer patients, i.e. when tumor cells disseminate to lymph nodes, further supports the putative immune evasive role of MGL ligands in metastatic disease.

The initial stages of template-controlled CaCO3 formation revealed by cryo-TEM.

Biogenic calcium carbonate forms the inorganic component of seashells, otoliths, and many marine skeletons, and its formation is directed by an ordered template of macromolecules. Classical nucleation theory considers crystal formation to occur from a critical nucleus formed by the assembly of ions from solution. Using cryotransmission electron microscopy, we found that template-directed calcium carbonate formation starts with the formation of prenucleation clusters. Their aggregation leads to the nucleation of amorphous nanoparticles in solution. These nanoparticles assemble at the template and, after reaching a critical size, develop dynamic crystalline domains, one of which is selectively stabilized by the template. Our findings have implications for template-directed mineral formation in biological as well as in synthetic systems.