Early detection of malignant tumours and, especially, micrometastases and disseminated tumour cells is still a challenge. In order to implement highly sensitive diagnostic tools we demonstrate the use of nanoprobes engineered from nanobodies (single-domain antibodies, sdAbs) and fluorescent quantum dots (QDs) for single- and two-photon detection and imaging of human micrometastases and disseminated tumour cells in ex vivo biological samples of breast and pancreatic metastatic tumour mouse models expressing human epidermal growth factor receptor 2 (HER2) or carcinoembryonic antigen (CEA). By staining thin (5–10 µm) paraffin and thick (50 µm) agarose tissue sections, we detected HER2- and CEA-positive human tumour cells infiltrating the surrounding tissues or metastasizing to different organs, including the brain, testis, lung, liver, and lymph nodes. Compared to conventional fluorescently labelled antibodies the sdAb-HER2-QD and sdAb-CEA-QD nanoprobes are superior in detecting micrometastases in tissue sections by lower photobleaching and higher brightness of fluorescence signals ensuring much better discrimination of positive signals versus background. Very high two-photon absorption cross-sections of QDs and small size of the nanoprobes ensure efficient imaging of thick tissue sections unattainable with conventional fluorescent probes. The nanobody–QD probes will help to improve early cancer diagnosis and prognosis of progression by assessing metastasis.

Fixation methods such as formalin are commonly used for the preservation of tissue with the aim of keeping their structure as close as possible to the native condition. However, fixatives chemically interact with tissue molecules, such as collagen in the extracellular matrix (ECM) or myosin, and may thus modify their structure. Taking advantage of the second- and third-harmonic generation (SHG and THG) emission capabilities of such components, we used nonlinear two-photon microscopy (NL2PM) to evaluate the effect that preservation methods, such as chemical fixatives, have on the nonlinear capabilities of protein components within mouse tissues. Our results show that depending on the preservation technique used, the nonlinear capabilities of collagen, lipid droplets and myosin microarchitecture are strongly affected. Parameters of collagen fibers, such as density and branch points, especially in collagen-sparse regions, e.g., in kidneys, were found to be altered upon formalin fixation. Moreover, cryo-freezing drastically reduced SHG signals from myosin. Our findings provide valuable information to select the best tissue fixation method for visualization and quantification of structural proteins, such as collagen and myosin by advanced NL2PM imaging techniques. This may advance the interpretation of the role these proteins play in disease.

The molecular pathogenesis of colorectal cancer is known to differ between the right and left side of the colon. Several previous studies have focussed on the differences in clinicopathological features, proteomic and genetic biomarkers, the composition of gut microbiota, response to therapy, and the characteristics of the tumour microenvironment. However, the morphology and density of collagen in the extracellular matrix (ECM) have not been studied intensively. In this study, we employed 2-photon laser scanning microscopy (2PLSM) to visualise the intrinsic second-harmonic generation (SHG) signal emitted by collagen fibres in the heterogeneous ECM of human colon tumour tissues. Through texture analysis of the SHG signal, we quantitatively distinguished the imaging features generated by structural differences of collagen fibres in healthy colon and cancers and found marked differences. The fibres inside of tumours exhibited a loss of organisation, particularly pronounced in right-sided colon cancer (RSCC), where the chaotic regions were significantly increased. In addition, a higher collagen content was found in left-sided colon cancer (LSCC). In future, this might aid in subclassification and therapeutic decisions or even in designing new therapy regimens by taking into account the differences between collagen fibres features between colon tumours located at different sides.

Background/Objectives: Immunotherapy has shown promising results in some cancers, but its efficacy remains limited in pancreatic ductal adenocarcinoma (PDAC). Vaccines in nanoparticle form (nanovaccines) can incorporate immunostimulating components to induce a potent immune response. As mesothelin (MSLN) is a tumor-associated antigen overexpressed in PDAC, we evaluated the effect of MSLN nanovaccine in a syngeneic orthotopic KPC-PDAC mouse model. Methods: An MSLN peptide combining three MSLN epitopes and two adjuvants, poly I:C and R848, was encapsulated in PLGA–chitosan nanoparticles to generate the nanovaccine. Results: The MSLN nanovaccine was successfully taken up by dendritic cells in vitro and was found in inguinal lymph nodes 24 h after subcutaneous injection into C57BL/6 mice. Nanovaccine re-stimulation of splenocytes from vaccinated mice led to increased levels of interferon-γ in vitro compared to unstimulated splenocytes. Higher levels of MSLN-specific IgM and IgG antibodies were detected in the serum of vaccinated mice compared to that of control mice. Three vaccination regimens were tested: a prophylactic scheme that included vaccination before tumor induction and two therapeutic schemes involving early and late vaccination after tumor cell inoculation. MSLN nanovaccination inhibited KPC tumor progression and metastasis and induced higher CD8⁺ T cell infiltration in the tumor that developed in response to prophylactic and early therapeutic schedules but not in response to a later vaccination approach. Although the nanovaccine treatment elicited higher humoral and cellular antigen-specific responses in tumor-bearing mice for both vaccination strategies, the therapeutic vaccination also increased the expression of exhaustion markers in CD8⁺ T cells. Conclusions: Our results support the relevance of an MSLN-based nanovaccine as a new immunotherapy treatment for PDAC and propose an innovative method of vaccine delivery using NPs.

Pancreatic ductal adenocarcinoma (PDAC) remains a major clinical challenge due to late detection and limited treatment responsiveness. To better evaluate complex immunotherapies in a human‐relevant setting, we developed an integrated organoid–immune co‐culture pipeline using PDAC patient‐derived organoids (PDOs) and matched HLA immune cells. As a proof of concept, we assessed an MSLN‐targeted nanovaccine (Mesovac), alone and in combination with FOLFIRINOX chemotherapy and Atezolizumab. We evaluated Mesovac across a multi‐stage pipeline, including T‐cell stimulation, ex vivo expansion, and PDO‐immune co‐cultures, to assess immune activation, specificity, and synergy with combinatorial treatments. MSLN‐stimulated T‐cells, derived from PDAC patients, showed increased IFN‐γ production and selective infiltration into MSLN‐expressing PDOs. Artificial antigen‐presenting cells (aAPCs) boosted the expansion of reactive T‐cells, enhancing antitumor responses. Notably, combining Mesovac with FOLFIRINOX and Atezolizumab maintained PD‐L1+ T‐cell levels and reduced cancer stem cells and aggressive PDAC subsets. Using this advanced in vitro workflow, we highlight that this platform, using human organoid–immune cell co‐cultures, enables the evaluation of complex processes related to nanovaccine strategies that would not be possible in vivo.